Synthetic and bio-hybrid nanoscale layers with tailored surface functionalities

We examine the prospective routes for the design of synthetic/biomacromolecular/inorganic film assemblies for photothermal cell based on biomimetic approach. We demonstrate examples of channel proteins immobilized onto surfaces of silicon single crystals modified with Langmuir–Blodgett and self-assembled monolayers. These proteins can be immobilized in intact, closed-pore conformation. Their state within photosensitive monolayers can be controlled by the photoisomerization reaction triggered by UV light.     

Ligand-directed immobilization of proteins through an esterase 2 fusion tag studied by atomic force microscopy

Atomically flat mica surfaces were chemically modified with an alkyl trifluoromethyl ketone, a covalent inhibitor of esterase 2 from Alicyclobacillus acidocaldarius, which served as a tag for ligand-directed immobilization of esterase-linked proteins. Purified NADH oxidase from Thermus thermophilus and human exportin-t from cell lysates were anchored on the modified surfaces. The immobilization effectiveness of the proteins was studied by atomic force microscopy (AFM). It was shown that ligand-esterase interaction allowed specific attachment of exportin-t and resulted in high-resolution images and coverage patterns that were comparable with immobilized purified protein. Moreover, the biological functionality of immobilized human exportin-t in forming a quaternary complex with tRNA and the GTPase Ran-GTP, and the dimension changes before and after complex formation were also determined by AFM.     

Surface modification of gold electrode with gold nanoparticles and mixed self-assembled monolayers for enzyme biosensors

This paper describes the development and evaluation of a generic method for the immobilization of enzymes onto a gold electrode and its application to amperometric biosensors. The surface of the gold electrode was modified with gold nano-particles (AuNP) and mixed self-assembled monolayers (SAMs) to form an enzyme biosensor matrix. Horseradish peroxidase (HRP) was immobilized on the modified surface to form a biosensor matrix on a gold electrode. After the deposition of gold nano-particles on a bare gold surface, the AuNP-deposited gold electrode and a bare electrode were compared for the surface area and electric current using AFM and cyclic voltammetry (CV). The AuNP strongly adhered to the surface of the gold electrode, had uniform distribution and was very stable. A mixed SAM, composed of two different monolayer molecules, dithiobis-N-succinimidyl propionate (DTSP) and inert tetradecane-1-thiol (TDT), was formed using reductive desorption technique and cyclic voltammetry was used to verify the formation of mixed deposition. First, 3-mercaptopropionic acid (MPA) and TDT were deposited with a specified deposition ratio between the two components. Then, MPA was desorbed by applying electric potential to the surface. Finally, DTSP was deposited where MPA was. The ratios of 20: 80 and 50: 50 between MPA and TDT were examined, and differences in the CV responses were discussed. HRP was immobilized on the mixed SAM surface. The investigated method is regarded as an effective way for stable enzyme attachment, while the presence of gold nanoparticles provides enhanced electrochemical activity; it needs very small amounts of samples and enzymes and the SAM matrix helps avoid enzyme leaking. It is interesting that the mixed SAM shows unique CV characteristics compared to the uni-molecular SAMs. The reaction kinetics of the SAM-immobilized enzyme is discussed with the CV results and is observed to obey the Michaelis-Menten equation.     

金属表面烷基硫醇类自组装膜的研究进展

自组装膜(SAMs)是活性分子通过化学键自发吸附在基底上的有序分子膜,由于其排列紧密、结构稳定,因此对基底金属具有良好的防腐蚀作用.本文综述了近年来烷基硫醇类自组装膜在金、铜、铁和其他金属表面缓蚀中的发展和现状.     

Electroreduction of laccase covalently bound to organothiol monolayers on gold electrodes

Cerrena unicolor laccase was immobilized on the gold electrode by covalent bonding to self-assembled monolayers of mercaptoundecanoic or mercaptopropionic acids. STM images of immobilized laccase proved high population of the laccase molecules on the monolayer modified electrode. The SERS experiments in concert with resonance Raman effect confirmed that the structure at the “blue” copper site of the immobilized protein remained intact. The accessibility of individual copper sites for electron exchange with the gold electrode surface was investigated by voltammetry. The electrode behavior of laccase is different in the presence and absence of oxygen, showing that the immobilized enzyme is reactive towards oxygen. Addition of two common mediators improved the electrical connectivity of the enzyme with the electrode, increased the catalytic efficiency of immobilized laccase and switched the onset of catalytic current to the potentials of the mediator. Immobilization of laccase on well-organized mercaptoundecanoic acid separates efficiently the enzyme from the electrode and does not allow easy access of mediators to the surface. Attachment of the enzyme at smaller distance from the electrode by means of significantly shorter spacer molecule—mercaptopropionic acid improved the efficiency of catalytic reduction of oxygen on the monolayer modified electrode.     

N-Hydroxysuccinimide-terminated self-assembled monolayers on gold for biomolecules immobilisation

Pure and mixed N-hydroxysuccinimide-terminated and butanethiol monolayers were prepared on flat gold (1 1 1) surfaces with the intent of developing suitable platforms for the direct biomolecules immobilisation. The self-assembled monolayers (SAMs) were characterised by electrochemical reductive desorption of the thiolate from the gold surface. The data have shown that certain solution proportions of the two compounds yield modified electrodes exhibiting intermediate electrochemical behaviour of the corresponding pure SAMs. The reactivity of the terminal N-hydroxysuccinimide (NHS) towards amine functionalities has been tested for the covalent attachment of Dopamine. The cyclic voltammetric responses of the investigated monolayers, after contacting with a Dopamine solution, have confirmed the chemical coupling of the amine as well as the formation of mixed SAMs. The Dopamine surface coverage increased with the amount of surface NHS. Laccase was also successfully immobilised onto this modified electrodes. The electrochemical behaviour of the modified SAMs with Laccase indicates direct electron transfer between the immobilised enzyme and the gold surface. Evidence for Laccase immobilisation was also provided by atomic force microscopic measurements.     

Ultrathin polypyrrole films on self-assembled monolayers as an efficient ultramicroelectrode assay

In this work, we investigated the effect of electrodeposition of polypyrrole (PPy) films on two different self-assembled monolayers (SAMs) modified gold electrode for the electrochemical construction of ultramicroelectrode (UME). In order to obtain SAM modified surfaces, 4-mercapto-1-butanol and 11-mercaptoundecanoic acid were used. The effect of these two chains on pyrrole electropolymerization was compared. Electropolymerization of pyrrole on SAM modified Au electrode was carried out by using cyclic voltammetry (CV) and constant potential electrolysis. To investigate the UME formation, the obtained surfaces were tested in Fe(CN)₆^3−/4− redox system. UMEs were characterized using scanning electron microscopy energy-dispersive X-ray spectroscopy, attenuated total reflectance fourier transform infrared spectroscopy and electrochemical impedance spectroscopy. The designed UME was applied as an immobilization matrix to entrap a redox protein, Hemoglobin (Hb), as a model. Direct electron transfer between this protein and the fabricated thiol modified PPy based UME was achieved, which is very challenging on bare electrode surfaces. With this study, a simple, low-cost and reproducible UME production way has been successfully accomplished and Hb modified UME is promising for different bioanalytical applications, for instance; cellular hydrogen peroxide or nitrite sensing.     

Formation of polyaniline layer on DNA by electrochemical polymerization

We report the electropolymerization and characterization of polyaniline (PAN) on both DNA monolayer and single poly(dG)–poly(dC) DNA molecules serving as a template. The synthesis includes the formation of an electrostatic complex between the negatively charged DNA and positively charged anilinium ions followed by electrochemical oxidation and polymerization of the anilinium ions' monomers on the DNA template. The polymerization was carried out on a flame annealed gold substrate modified with positively charged self-assembled 4-aminothiophenol (4-ATPh) monolayer. The resulting monolayers and single macromolecular composites of DNA–PAN were characterized by atomic force microscopy (AFM), cyclic voltammetry (CV) impedance and UV–vis spectroscopy.     

Surface chemistry and electrocatalytic behaviour of tetra-carboxy substituted iron, cobalt and manganese phthalocyanine monolayers on gold electrode

Surface chemistry and electrocatalytic properties of self-assembled monolayers of metal tetra-carboxylic acid phthalocyanine complexes with cobalt (Co), iron (Fe) and manganese (Mn) as central metal ions have been studied. These phthalocyanine molecules are immobilized on gold electrode via the coupling reaction between the ring substituents and pre-formed mercaptoethanol self-assembled monolayer (Au-ME SAM). X-ray photoelectron spectroscopy (XPS) and Raman spectroscopy confirmed chemisorption of mercaptoethanol via sulfur group on gold electrode and also coupling reaction between phthalocyanines and Au-ME SAM. Electrochemical parameters of the immobilized molecules show that these molecules are densely packed with a perpendicular orientation. The potential applications of the gold modified electrodes were investigated towards l-cysteine detection and the analysis at phthalocyanine SAMs. Cobalt and iron tetra-carboxylic acid phthalocyanine monolayers showed good oxidation peak for l-cysteine at potentials where metal oxidation (M^III/M^II) takes place and this metal oxidation mediates the catalytic oxidation of l-cysteine. Manganese tetra-carboxylic acid phthalocyanine monolayer also exhibited a good catalytic oxidation peak towards l-cysteine at potentials where Mn^IV/Mn^III redox peak occurs and this redox peak mediates l-cysteine oxidation. The analysis of cysteine at phthalocyanine monolayers displayed good analytical parameters with good detection limits of the orders of 10⁻⁷ mol L⁻¹ and good linearity for a studied concentration range up to 60 μmol L⁻¹.     

Patterns of functional proteins formed by local electrochemical desorption of self-assembled monolayers

Patterned self-assembled monolayers (SAMs) were formed using the scanning electrochemical microscope (SECM). The procedures is based on the local electrochemical desorption of an alkanethiolate monolayer by applying a 5 kHz square-wave voltage of 2 V (peak-to-peak) to a two-electrode configuration consisting of an ultramicroelectrode (UME) of 10 μm diameter placed about 5 μm above a macroscopic SAM-covered gold electrode. Desorption occurs on well-defined regions with a diameter of (12.8±2.8) μm. These regions of bare gold are able to chemisorb a ω-functionalized thiol or disulfide such as cystamine to form patterns of amino-terminated surfaces. Functional proteins can be coupled to the amino groups present at the modified regions of the monolayer. This approach was demonstrated by imaging the activity of horseradish peroxidase bound to the patterned SAMs in the generation–collection mode of the SECM. A considerable improvement of the procedure could be achieved by performing the desorption in a solution containing a millimolar concentration of the ω-functionalized thiol/disulfide ensuring effective refilling of the monolayer by the desired molecules and hence high concentration of the immobilized proteins. The method is discussed with respect to prospective application in the field of chip-based bioanalytical assays.     

Preparation and Characterization of Covalently Binding of Rat Anti-human IgG Monolayer on Thiol-Modified Gold Surface

The 16-mercaptohexadecanoic acid (MHA) film and rat anti-human IgG protein monolayer were fabricated on gold substrates using self-assembled monolayer (SAM) method. The surface properties of the bare gold substrate, the MHA film and the protein monolayer were characterized by contact angle measurements, atomic force microscopy (AFM), grazing incidence X-ray diffraction (GIXRD) method and X-ray photoelectron spectroscopy, respectively. The contact angles of the MHA film and the protein monolayer were 18° and 12°, respectively, all being hydrophilic. AFM images show dissimilar topographic nanostructures between different surfaces, and the thickness of the MHA film and the protein monolayer was estimated to be 1.51 and 5.53 nm, respectively. The GIXRD 2θ degrees of the MHA film and the protein monolayer ranged from 0° to 15°, significantly smaller than that of the bare gold surface, but the MHA film and the protein monolayer displayed very different profiles and distributions of their diffraction peaks. Moreover, the spectra of binding energy measured from these different surfaces could be well fitted with either Au4f, S2p or N1s, respectively. Taken together, these results indicate that MHA film and protein monolayer were successfully formed with homogeneous surfaces, and thus demonstrate that the SAM method is a reliable technique for fabricating protein monolayer.     

Polystyrene-based diazonium salt as adhesive: A new approach for enzyme immobilization on polymeric supports

In this work, a new way for enzyme immobilization was explored and properties of the enzyme immobilized on different polymer films were investigated. In the process, a polystyrene-based diazonium salt (PS-DAS) was synthesized and used as molecular adhesive to immobilize β-glucosidase on the polymeric supports (films of polyethylene, polypropylene and poly(ethylene terephthalate)). The immobilization of β-glucosidase on the polymer surfaces was achieved by sequential depositions of a piece of the polymer films in PS-DAS and the enzyme solutions. The surface modification was investigated by X-ray photoelectron spectroscopy (XPS), water contact angle measurement, and atomic force microscopy (AFM). The activity of the immobilized β-glucosidase was evaluated by measuring its enzymatic activity to the hydrolysis of p-nitrophenyl-β-d-glucopyranoside (pNPG). The optimized reaction conditions (such as pH and temperature), thermal stability, and reusability of the immobilized enzyme on PE films were assayed by using the enzyme-catalyzed reaction. Results show that the polymeric diazonium salt is firmly adhered on the polymer surfaces and the modified surfaces can react with the enzyme to form covalent bonds. The immobilized enzyme shows changes in the optimized pH and temperature for the hydrolysis reaction catalyzed by β-glucosidase. The kinetic parameter (K_m) of the immobilized β-glucosidase is lower than that of its free counterpart. The immobilized enzyme shows significant enhancement in the thermal stability and reasonable reusability. This new approach can be used as a simple and versatile method for protein immobilization.     

A stability comparison of redox-active layers produced by chemical coupling of an osmium redox complex to pre-functionalized gold and carbon electrodes

The production of stable redox active layers on electrode surfaces is a key factor for the development of practical electronic and electrochemical devices. Here, we report on a comparison of the stability of redox layers formed by covalently coupling an osmium redox complex to pre-functionalized gold and graphite electrode surfaces. Pre-treatment of gold and graphite electrodes to provide surface carboxylic acid groups is achieved via classical thiolate self-assembled monolayer formation on gold surfaces and the electro-reduction of an in situ generated aryldiazonium salt from 4-aminobenzoic acid on gold, glassy carbon and graphite surfaces. These surfaces have been characterized by AFM and electrochemical blocking studies. The surface carboxylate is then used to tether an osmium complex, [Os(2,2′-bipyridyl)₂(4-aminomethylpyridine)Cl]PF₆, to provide a covalently bound redox active layer, E^0′ of 0.29 V (vs. Ag/AgCl in phosphate buffer, pH 7.4), on the pre-treated electrodes. The aryldiazonium salt-treated carbon-based surfaces showed the greatest stability, represented by a decrease of <5% in the peak current for the Os(II/III) redox transition of the immobilized complex over a 3-day period, compared to a decrease of 19% and 14% for the aryldiazonium salt treated and thiolate treated gold surfaces, respectively, over the same period.     

Preparation of protein microarrays on non‐fouling and hydrated poly(ethylene glycol) hydrogel substrates using photochemical surface modification

BACKGROUND: Conventional protein microarrays prepared on hard, dry substrates, such as glass and silicone, have several limitations, as proteins may easily denature and lose their structure. To overcome such problems, the fabrication of wet protein microarrays on non‐fouling and hydrated PEG‐based hydrogels was investigated. RESULT: Bovine serum albumin (BSA) and glucose oxidase (GOX), chosen as model proteins, were covalently immobilized on PEG hydrogel surfaces via 5‐azidonitrobenzoyloxy N‐hydroxysuccinimide, a photoreactive bifunctional linker. Successful fixation of the bifunctional linker and subsequent immobilization of the proteins on the PEG hydrogel surfaces were confirmed with X‐ray photoelectron spectroscopy (XPS) and Fourier transform infrared spectroscopy (FTIR) studies. GOX immobilized on the hydrogel surface maintained approximately 50% of its initial activity after 24 h when left in dry conditions, but maintained only 20% when immobilized on a dry substrate. Photochemical fixation combined with photolithography produced well‐defined protein micropatterns with sizes ranging from 50–500 µm, and molecular recognition‐mediated specific binding between biotin and streptavidin was successfully assayed using microarrays on PEG hydrogels. CONCLUSION: A protein‐repellent PEG hydrogel surface was photochemically modified to covalently immobilize proteins and create protein microarrays. The use of hydrated hydrogels as substrates for protein microarrays could minimize the deactivation of proteins in dry conditions, and the non‐fouling property of PEG hydrogels allows the passivation step of protein microarray preparation to be skipped. Copyright © 2008 Society of Chemical Industry     

Self-assembling functionalized templates in biosensor technology

A bifacial topological template exhibiting metal-binding sites and thioalkane chains has been incorporated into self-assembled monolayers and immobilised on gold surfaces: These systems allow for the detection of external ligands by SPR spectroscopy representing a first step in developing biosensors based on the TASP (Template Assembled Synthetic Proteins) concept. Received: 17 November 1997/Accepted: 12 December 1997     

Preparation of 7-azidocarbonyl-2,4,9-trithiaadamantane by a new thioacetal crown synthetic method

A new method for synthesizing thioacetal crown is reported for the effective synthesis of 7-methoxycarbonyl-2,4,9-trithiaadamantane. Application of this intermediate in the synthesis of 7-azidocarbonyl-2,4,9-trithiaadamantane, a photoreactive surface linker for photolithographic chemical patterning on self-assembled monolayers on gold surfaces, is also reported.     

Atomic force microscopy (AFM) characterisation of the porous silica nanostructure of two centric diatoms

The porous silica nanostructure of two marine, centric diatoms, Coscinodiscus sp. and Thalassiosira eccentrica was investigated by atomic force microscopy (AFM). Important morphological features of the silica frustules of diatoms are described, including: the organisation of porous silica layers, their topography, pore size, shape and density. The outer layer of Coscinodiscus sp., commonly called the cribellum, consists of a characteristic hexagonal array of pores with pore sizes of around 45 nm. This thin membrane covers a second structural layer where two different silica surfaces are identified. The outer part, known as the cribrum consists of hexagonally packed pores of about 200 nm diameter. The inner part, known as the foramen layer, consists of larger and radially distributed holes with a diameter of around 1,150 nm. The second diatom species investigated, T. eccentrica produces a frustule with one silica structural layer featuring two different porous surfaces. The outer surface has large (800 nm diameter) holes (foramen) while the inner surface contains a porous wall with pores comparable in size to the Coscinodiscus sp. cribellum. The inner and outer surfaces of the frustule wall of both diatoms are hence in reverse order. However, the size of the small pores is similar for both species. High-resolution AFM also revealed the granular nanostructure of the diatom biosilica with grain sizes from 20 to 70 nm diameters.     

One step gold (bio)functionalisation based on CS2-amine reaction

Dithiocarbamates have been regarded as alternative anchor groups to thiols on gold surfaces, and claimed to be formed in situ through the reaction between secondary amines and carbon disulphide. In this paper, we further exploit this methodology for a convenient one step biomolecule immobilisation onto gold surfaces. First, the reactivity between CS₂ and electroactive compounds containing amines, primary (dopamine), secondary (epinephrine), and an amino acid (tryptophan) has been investigated by electrochemical methods. Cyclic voltammetric characterisation of the modified electrodes confirmed the immobilisation of all the target compounds, allowing the estimation of their surface concentration. The best result was obtained with epinephrine, a secondary amine, for which a typical quasi-reversible behaviour of surface confined electroactive species could be clearly depicted. Electrochemical reductive desorption studies enabled to infer on the extent of the reaction and on the relative stability of the generated monolayers. Bio-functionalisation studies have been accomplished through the reaction of CS₂ with glucose oxidase in aqueous medium, and the catalytic activity of the immobilised enzyme was evaluated towards glucose, by electrochemical methods in the presence of a redox mediator. Scanning tunnelling microscopy (STM) and Atomic force microscopy (AFM) were used respectively, to characterize the gold electrodes and Glucose Oxidase coverage and distribution on the modified surfaces.     

The Impervious Route to Peptide-Based Dye-Sensitized Solar Cells

Electron transfer through peptide scaffolds has been studied for a long time as a useful and simple approach to investigate the mechanisms of the ubiquitous electron-transfer processes in biology. Here, we review our long-standing work in the field, focused on the study of the electronic conduction properties of hybrid devices based on peptide films immobilized on metal surfaces. In particular, we describe the electrochemical and photoelectrochemical properties of peptide-based self-assembled monolayers chemically linked to gold substrates through a disulfide group. Very recently, this work culminated in the construction of a prototype dye-sensitized solar cell, the photoactive element of which was constituted by a helical oligopeptide functionalized with a near-UV-absorbing chromophore. We also take advantage of the occasion to critically review the state of the art of the field, with suggestions for the issues needing further investigation.     

Covalent crowding strategy for trypsin confined in accessible mesopores with enhanced catalytic property and stability

Chemically modified macromolecules were assembled with adsorptive trypsin in mesoporous silica foams (MCFs) to establish covalent linkage. Effects of catalytic properties and stability of immobilized trypsin were examined. The addition of chemically modified protein (BSA) and polysaccharide (ficoll) to the immobilized trypsin exhibited high coupled yield (above 90%) and relative activities (174.5% and 175.9%, respectively), showing no protein leaching after incubating for 10 h in buffers. They showed broader pH and temperature profiles, while the half life of thermal stability of BSA-modified preparation at 50 °C increased to 1.3 and 2.3 times of unmodified and free trypsin, respectively. The modified trypsin in aqueous-organic solvents exhibited 100% activity after 6 h at 50 °C. The kinetic parameters of trypsin preparations and suitable pore diameter of MCFs warranted compatibility of covalent modification for substrate transmission. The covalent crowding modification for immobilized trypsin in nanopores establishes suitable and accessible microenvironment and renders possibility of biological application.