Structural control of surface layer proteins at electrified interfaces investigated by in situ Fourier transform infrared spectroscopy

In situ Fourier Transform Infrared (FTIR) Spectroscopy complemented by Electrochemical Quartz Microbalance (EQMB) investigations allowed a detailed insight into the influence of the electrode potential on competing adsorption processes and bonding mechanisms of buffer ions and S-layer protein molecules of Lysinibacillus sphaericus CCM2177 at an electrified liquid/gold interface. The S-layer proteins adsorb on gold polarized positively of the point of zero charge by displacing perchlorate anions in the Helmholtz plane by their carboxylate groups. This is indicated by an increase of the peptide and carboxylate infrared absorption signals accompanied by a decrease of the perchlorate signal. S-layers interlinked laterally with Ca(2+) ions, positive of the point of zero charge, resulted in the formation of a crystalline layer participating in the Helmholtz layer. In contrast to the absence of the Ca(2+)-linkers, S-layers remain structurally intact also in the negative polarization domain where the Helmholtz layer is solely sustained by mainly solvated cations without participation of the negatively charged protein carboxylate functions.     

Characterization and use of crystalline bacterial cell surface layers

Crystalline bacterial cell surface layers (S-layers) are one of the most common outermost cell envelope components of prokaryotic organisms (archaea and bacteria). S-layers are monomolecular arrays composed of a single protein or glycoprotein species and represent the simplest biological membranes developed during evolution. S-layers as the most abundant of prokaryotic cellular proteins are appealing model systems for studying the structure, synthesis, genetics, assembly and function of proteinaceous supramolecular structures. The wealth of information existing on the general principle of S-layers have revealed a broad application potential. The most relevant features exploited in applied S-layer research are: (i) pores passing through S-layers show identical size and morphology and are in the range of ultrafiltration membranes; (ii) functional groups on the surface and in the pores are aligned in well-defined positions and orientations and accessible for chemical modifications and binding functional molecules in very precise fashion; (iii) isolated S-layer subunits from a variety of organisms are capable of recrystallizing as closed monolayers onto solid supports (e.g., metals, polymers, silicon wafers) at the air–water interface, on lipid films or onto the surface of liposomes; (iv) functional domains can be incorporated in S-layer proteins by genetic engineering. Thus, S-layer technologies particularly provide new approaches for biotechnology, biomimetics, molecular nanotechnology, nanopatterning of surfaces and formation of ordered arrays of metal clusters or nanoparticles as required for nanoelectronics.     

Influence of surface chemistry and protein concentration on the adsorption rate and S-layer crystal formation

Bacterial crystalline surface layers (S-layers) are the outermost envelope of prokaryotic organisms representing the simplest biological membranes developed during evolution. In this context, the bacterial protein SbpA has already shown its intrinsic ability to reassemble on different substrates forming protein crystals of square lattice symmetry. In this work, we present the interaction between the bacterial protein SbpA and five self-assembled monolayers carrying methyl (CH(3)), hydroxyl (OH), carboxylic acid (COOH) and mannose (C(6)H(12)O(6)) as functional groups. Protein adsorption and S-layer formation have been characterized by atomic force microscopy (AFM) while protein adsorption kinetics, mass uptake and the protein layer viscoelastic properties were investigated with quartz crystal microbalance with dissipation monitoring (QCM-D). The results indicate that the protein adsorption rate and crystalline domain area depend on surface chemistry and protein concentration. Furthermore, electrostatic interactions tune different protein rate adsorption and S-layer recrystallization pathways. Electrostatic interactions induce faster adsorption rate than hydrophobic or hydrophilic interactions. Finally, the shear modulus and the viscosity of the recrystallized S-layer on CH(3)C(6)S, CH(3)C(11)S and COOHC(11)S substrates were calculated from QCM-D measurements. Protein-protein interactions seem to play a main role in the mechanical stability of the formed protein (crystal) bilayer.     

An Amperometric Glucose Sensor Based on Isoporous Crystalline Protein Membranes as Immobilization Matrix

Abstract

S-layer ultrafiltration membranes (SUMs) with an active filtration layer composed of coherent two-dimensional, isoporous protein crystals (S-layers) have been used as matrix for immobilizing monolayers of enzymes. Since S-layers are formed by periodic repetition of identical protein subunits, functional groups are present on the crystalline array in an identical position and orientation. As a consequence monolayers of enzymes can bind in a geometrically well defined way. For the covalent immobilization of enzymes carboxyl groups from the S-layer protein were activated with carbodiimide and allowed to react with amino groups of the enzyme. SUMs were employed as a new type of immobilization matrix for the developement of an amperometric glucose sensor using glucose oxidase (GOD) as the biologically active component. Glucose oxidase covalently bound to the surface of the S-layer protein retained approximately 40% of its activity. The enzyme loaded SUMs were covered with a layer of gold or platinum to function as working electrodes. These sensors yielded high signals (150nA/mm²/mmol glucose), fast response times (10–30s) and a linearity range up to 12 mM glucose. The stability under working conditions was more than 48 hours. There was no loss in activity after a storage period of 6 month.     

Thin film processing using S-layer proteins: biotemplated assembly of colloidal gold etch masks for fabrication of silicon nanopillar arrays

We explored the bionanofabrication of silicon nanopillar structures using ordered gold nanoparticle arrays generated from microbial surface layer (S-layer) protein templates. The S-layer template used for these thin film processing experiments was isolated from the Gram-positive bacterium Deinococcus radiodurans. In this preliminary work, S-layers preimmobilized onto chemically modified silicon substrates were initially used to template the fabrication of a nanolithographic hard mask pattern comprised of a hexagonally ordered array of 5-nm gold nanoparticles (lattice constant = 18 nm). Significantly, the use of the biotemplated gold nanoparticle mask patterns in an inductively coupled plasma (ICP) etching process successfully yielded silicon nanopillar structures. However, it was found that the resultant nanopillars (8–13 nm wide at the tip, 15–20 nm wide at half-height, 20–30 nm wide at the base, and 60–90 nm tall) appeared to lack any significant degree of translational ordering. The results suggest that further studies are needed in order to elucidate the optimal plasma processing parameters that will lead to the generation of long-range ordered arrays of silicon-based nanostructures using S-layer protein templates.     

In-situ infrared study of the adsorption and oxidation of oxalic acid at single-crystal and thin-film gold electrodes: a combined external reflection infrared and ATR-SEIRAS approach

The adsorption and oxidation of oxalic acid at gold electrodes were studied by in-situ infrared spectroscopy. External reflection experiments carried out with gold single-crystal electrodes were combined with internal reflection (ATR-SEIRAS) experiments with gold thin-film electrodes. These gold thin films, with a typical thickness of ca. 35 nm, were deposited on silicon substrates by argon sputtering. As previously reported for evaporated gold films, the voltammetric curves obtained in sulfuric acid solutions after electrochemical annealing show typical features related to the presence of wide bidimensional (111) domains with long-range order. The in-situ infrared data collected for solutions of pH 1 confirmed the potential-dependent adsorption of either oxalate (Au(100)) or a mixture of bioxalate and oxalate (Au(111), Au(110), and gold thin films) anions in a bidentate configuration. The better signal-to-noise ratio associated with the SEIRA effect in the case of the gold thin-film electrodes allows the observation of the carbonyl band for adsorbed bioxalate that was not detected in the external reflection experiments. Besides, additional bands are observed between 2000 and 3000 cm(-)(1) that can be tentatively related to the formation of hydrogen bonds between neighboring bioxalate anions. The intensities of these bands decrease with increasing solution pH values, disappearing for pH 3 solutions in which adsorbed oxalate anions are the predominant species. The analysis of the intensities of the nu(s)(O-C-O) and nu(C-OH) + delta(C-O-H) bands for adsorbed oxalate and bioxalate, respectively, suggests that the pK(a) for the surface equilibrium between these species is significantly lower than that for the solution equilibrium.     

A Multistep Enzyme Sensor for Sucrose Based on S-Layer Microparticles As Immobilization Matrix

Abstract

In this paper we report on the construction principle and performance of an amperometric 3-enzyme sensor for sucrose based on crystalline bacterial cell surface layers (S-layers) as immobilization matrix for the biological components.

Isoporous, crystalline surface layers (S-layers) have been identified as outermost cell envelope layer in many bacteria. Since they are composed of identical protein or glycoprotein subunits with functional groups in well defined positions and orientations, they represent ideal matrices for the controlled and reproducible immobilization of functional macromolecules, as required for the development of biosensors. Apart from single enzyme sensors, which were described earlier, a strikingly simple method for the assembly and optimization of multistep systems was developed. For the fabrication of an amperometric sucrose sensor invertase, mutarotase and glucose oxidase were individually immobilized on S-layer fragments isolated from Clostridium thermohydrosulfuricum L111-69 via aspartic acid as spacer molecules. Subsequently, appropriate mixtures of enzyme loaded S-layer fragments were deposited on a microfiltration membrane and finally, the composite multifunctional sensing layer was sputtered with gold in order to establish a good metal contact. Amperometric sucrose measurements based on H₂O₂ oxidation revealed a high signal level (1 μA^?1/cm^2?mmol sucrose), 5 min response time and a linear range up to 30 mM sucrose as the main characteristics of the S-layer sucrose sensor.     

Self-assembly of S-layer-enveloped cytochrome c polyelectrolyte multilayers

We report a study of the electrostatic layer-by-layer self-assembly of electroactive polyelectrolyte multilayers incorporating the redox protein cytochrome c (cyt c) combined with recrystallization of the bacterial cell wall surface layer from Bacillus sphaericus CCM 2177 SbpA (S-layer). The polyelectrolyte multilayer assembly was prepared on flat gold electrodes with a nanometer-scale roughness that allowed monitoring of the film formation throughout all the assembly stages by atomic force microscopy measurements in liquid with respect to topography and forces. The deposition of alternating layers of sulfonated polyaniline and cyt c was carried out by adsorption from the corresponding solutions on a cyt c monolayer electrode. The electroactivity of cyt c within the assembly was confirmed by cyclic voltammetry. We showed that the surface properties of the electrode terminating layer change after each adsorption step accordingly. We also found that S-layer recrystallization on the top of the multilayer film was feasible while electroactivity of cyt c within a polyelectrolyte matrix was partially maintained. This approach offers a new strategy to design a biocompatible and permselective outer envelope of a polyelectrolyte multilayer, promising sensor applications.     

Formation of a gold superlattice on an S-layer with square lattice symmetry

This work describes a new strategy in which a crystalline bacterial cell surface layer (S-layer) composed of a monolayer of a single protein species was used as periodic nanometric template in the nucleation of ordered arrays of gold nanoparticles. A square superlattice of uniform 4 to 5 nm sized gold particles with 12.8 nm repeat distance was fabricated by exposing the S-layer lattice of Bacillus sphaericus CCM2177, in which thiol groups had been introduced before, to a tetrachloroauric(III) acid solution. Transmission electron microscopical studies showed that the gold nanoparticles were formed in the pore region during electron irradiation of an initially grainy gold coating covering the whole S-layer lattice. The shape of the gold particles resembled the morphology of the pore region of the square S-layer lattice. By electron diffraction and energy dispersive X-ray analysis the crystallites were identified as gold (Au(0)). Electron diffraction patterns revealed that the gold nanoparticles were crystalline but in the long range order not crystallographically aligned. It is postulated that S-layers will allow the fabrication of a wide range of inorganic nanocrystal superlattice arrays.     

Probing the protein orientation on charged self-assembled monolayers on gold nanohole arrays by SERS

In this work, surface-enhanced Raman scattering (SERS) was applied to probe the orientation of cytochrome c (Cyt-c) on gold nanohole arrays functionalized with self-assembled monolayers (SAMs) of alkane thiols with positively (-NH2) and negatively (-COOH) charged terminal groups. Square grid gold nanohole arrays with a nanohole diameter of 270 nm and a grating of 350 nm were fabricated by electron beam lithography (EBL) and were used as the SERS substrates. The SERS intensities of the nontotally symmetric mode (B(1g) mode nu(11)) and the totally symmetric mode (A(1g) mode nu(4)) and their ratios were used to determine the orientation of Cyt-c on surfaces. The results indicate that the heme group is close and perpendicular to the negatively charged surface but is far from and oriented at an angle to the positively charged surface. Cyt-c has a random or more flat orientation on the bare Au nanoholes surface.     

Ultrafiltration membranes prepared from crystalline bacterial cell surface layers as model systems for studying the influence of surface properties on protein adsorption

Crystalline bacterial cell surface layers (S-layers) were used for the preparation of the active filtration layer of ultrafiltration membranes (S-layer ultrafiltration membranes; SUMs). Since the S-layer is uniform in its pore size and morphology and its functional groups are aligned in well-defined positions, the SUMs provide ideal model systems for studying protein adsorption and membrane fouling. Due to the presence of surface-located carboxyl groups the standard SUMs have the net negative charge but exhibit basically a hydrophobic character. In order to change the net charge, the charge density and the accessibility of charged groups of the SUMs as well as their hydrophobicity, free carboxyl groups of the S-layer protein were modified with selected low molecular weight nucleophiles under conditions of preserving the crystalline lattice structure. SUMs with 1.6 to 7 charged or functional groups exposed per nm² of the membrane area were used for adsorption experiments. After solutions of differently sized and charged test proteins were filtered, the relative flux losses of distilled particle free water were measured. The results showed that the adsorption capacity of the SUMs increased with the extent of their hydrophobicity. Test proteins showed their own specific adsorption characteristics, which clearly demonstrated the difficulties in determining parameters controlling the membrane fouling. Independent of the net charge of the test proteins and that of the SUMs, the flux loss of SUMs increased with the increased charge density and an improved accessibility of the charged groups on the S-layer surface. No essential differences in the adsorption characteristics were observed between the zwitterionic SUMs of slightly surplus of free carboxyl groups and the standard SUMs of net negative charge.     

Electrostatic attachment of gold and poly(lactic acid) nanoparticles onto omega-aminoalkanoic acid self-assembled monolayers on 316L stainless steel

The assembly of poly(lactic acid) (PLA) nanoparticles on a 12-aminodecanoic acid (ADA) self-assembled monolayer (SAM) is described. Assembly is accomplished through electrostatic interactions between the positively charged SAM and the negatively charged PLA nanoparticles. The strategy used involves two steps in which a preliminary electrochemical coating of the ADA SAM is followed by a second step that involves immersing the SAM in a solution containing gold or PLA nanoparticles. The SAM was characterized by using cyclic voltammetry (CV), X-ray photoelectron spectroscopy (XPS), FTIR spectroscopy, and contact angle measurements, whereas scanning electron microscopy (SEM) was used to image the nanoparticles after electrostatic attachment was achieved. We found that the surface coverage of the nanoparticles could be controlled by modulating the electrostatic interactions between the negatively charged particles and the positively charged SAM surface by varying the pH of the nanoparticle solution, the immersion time, and the number of cyclic voltammetry scans under which the SAM was formed.     

Model electrodes with defined mesoscopic structure

Model electrodes with defined mesoscopic structure were either generated by adsorption of surfactant stabilized metal clusters from colloidal solution on a support of gold or by electrochemical deposition of platinum on gold substrates. Both types of model electrodes were characterized by STM (scanning tunnelling microscopy), cyclic voltammetry and electrooxidation of adsorbed CO. The supported colloidal Pt as well as the electrochemically deposited Pt revealed different reactivities regarding the CO monolayer electrooxidation as compared to a polycrystalline Pt bulk electrode. In addition, in-situ FTIR (Fourier transformed infrared) spectroscopy was applied to characterize CO adsorbed on electrochemically deposited Pt on gold. Combined with the structural information from STM it seems likely that the differences regarding the catalytic properties of the model electrodes are due to different coverages of the substrate with catalyst particles. Received: 24 June 1996 / Revised: 29 November 1996 / Accepted: 4 December 1996     

Electrocatalytic oxidation of reduced nicotinamide adenine dinucleotide (NADH) at thick-film gold electrodes

Cyclic voltammetric and electrochemical impedance spectroscopic investigations of screen-printed, thick-film gold electrodes reveal significant differences when compared with conventional polished gold disk electrodes of comparable size. The rough and porous structure of the thick-film electrode surface leads to an actual electrode area which is increased six-fold compared to polished disk electrodes. Due to the catalytic properties of these surface structures it is possible to perform the electrochemical oxidation of reduced nicotinamide adenine dinucleotide (NADH) at relatively low overpotentials, i.e. +0.145 V vs. SCE. By operating electrodes at this potential, electrode fouling processes and interference from electroactive species, e.g. acetaminophen, are minimized. An amperometric glucose sensor based on polymer matrix-entrapped glucose dehydrogenase with a working potential of +0.145 V vs. SCE was successfully incorporated into a flow injection analysis (FIA) system.     

Kappa-casein based electrochemical and surface plasmon resonance biosensors for the assessment of the clotting activity of rennet

We report for the first time the development of kappa-casein (κ-CN)-based electrochemical and surface plasmon resonance (SPR) biosensors for the assessment of the clotting activity of rennet. Electrochemical biosensors were developed over gold electrodes modified with a self-assembled monolayer of dithiobis-N-succinimidyl propionate, while SPR measurements were performed on regenerated carboxymethylated dextran gold surfaces. In both types of biosensor, κ-CN molecules were immobilized onto modified gold surfaces through covalent bonding. In electrochemical biosensors, interactions between the immobilized κ-CN molecules and chymosin (the active component of rennet) were studied by performing cyclic voltammetry, differential pulsed voltammetry, and electrochemical impedance spectroscopy (EIS) measurements, using hexacyanoferrate(II)/(III) couple as a redox probe. κ-CN is cleaved by rennet at the Phe105–Met106 bond, producing a soluble glycomacropeptide, which is released to the electrolyte, and the positively charged insoluble para-κ-casein molecule, which remains attached to the surface of the electrode. This induced reduction of the net negative charge of the sensing surface, along with the partial degradation of the sensing layer, results in an increase of the flux of the redox probe, which exists in the solution, and consequently, to signal variations, which are associated with the increased electrocatalysis of the hexacyanoferrate(II)/(III) couple on the gold surface. SPR experiments were performed in the absence of the redox probe and the observed SPR angle alterations were solely attributed to the cleavage of the immobilized κ-CN molecules. Various experimental variables were investigated and under the selected conditions the proposed biosensors were successfully tried to real samples. The ratios of the clotting power units in various commercial solid or liquid samples, as they are calculated by the EIS-based data, were almost identical to those obtained with a reference method. In addition, EIS measurements showed an excellent reproducibility, lower than 5%.     

Investigation of the electrochemical and electrocatalytic behavior of positively charged gold nanoparticle and L-cysteine film on an Au electrode

Positively charged gold nanoparticle (positively charged nano-Au), which was prepared, characterized by ξ-potential and transmission electron microscopy (TEM) was used in combination with l-cysteine to fabricate a modified electrode for electrocatalytic reaction of biomolecules. Compared with electrodes modified by negatively charged gold nanoparticle/l-cysteine, or l-cysteine alone, the electrode modified by the positively charged gold nanoparticle/l-cysteine exhibited excellent electrochemical behavior toward the oxidation of biomolecules such as ascorbic acid, dopamine and hydrogen peroxide. Moreover, the proposed mechanism for electrocatalytic response of positively charged gold nanoparticle was discussed. The immunosensor showed a specific to ascorbic acid in the range 5.1 × 10⁻⁷-6.7 × 10⁻⁴ M and a low detection limit of 1.5 × 10⁻⁷ M. The experimental results demonstrate that positively charged gold nanoparticle have more efficient electrocatalytic reaction than negatively charged gold nanoparticle, which opens up new approach for fabricating sensor.     

Prolonged stochastic single ion channel recordings in S-layer protein stabilized lipid bilayer membranes

S-layer proteins are commonly found in bacteria and archaea as two-dimensional monomolecular crystalline arrays as the outermost cell membrane component. These proteins have the unique property that following disruption by chemical agents, monomers of the protein can re-assemble to their original lattice structure. This unique property makes S-layers interesting for utilization in bio-nanotechnological applications. Here, we show that the addition of S-layer proteins to bilayer lipid membranes increases the lifetime and the stability of the bilayer. M2delta ion channels were functionally incorporated into these S-layer stabilized membranes and we were able to record their activity for up to 20 h. Transmission electron microscopy (TEM) was used to visualize the 2D crystalline pattern of the S-layer and the M2delta ion channel characteristics in bilayer lipid membrane's were compared in the presence and absence of S-layers.     

Bionanofabrication of metallic and semiconductor nanoparticle arrays using S-layer protein lattices with different lateral spacings and geometries

Two-dimensional (2-D) surface layer (S-layer) protein lattices isolated from the gram-positive bacterium Deinococcus radiodurans and the acidothermophilic archaeon Sulfolobus acidocaldarius were investigated and compared for their ability to biotemplate the formation of self-assembled, ordered arrays of inorganic nanoparticles (NPs). The NPs employed for these studies included citrate-capped gold NPs and various species of CdSe/ZnS core/shell quantum dots (QDs). The QD nanocrystals were functionalized with different types of thiol ligands (negative- or positive-charged/short- or long-chain length) in order to render them hydrophilic and thus water-soluble. Transmission electron microscopy, Fourier transform analyses, and pair correlation function calculations revealed that ordered nanostructured arrays with a range of spacings (approximately 7-22 nm) and different geometrical arrangements could be fabricated through the use of the two types of S-layers. These results demonstrate that it is possible to exploit the physicochemical/structural diversity of prokaryotic S-layer scaffolds to vary the morphological patterning of nanoscale metallic and semiconductor NP arrays.     

Nanohybrid Materials Consisting of Poly[(3‐aminobenzoic acid)‐co‐(3‐aminobenzenesulfonic acid)‐co‐aniline] and Multiwalled Carbon Nanotubes for Immobilization of Redox Active Cytochrome c

The development of a new surface architecture for the efficient direct electron transfer of positively charged redox proteins is presented. For this reason different kinds of polyaniline terpolymers consisting of aminobenzoic acid (AB), aminobenzenesulfonic acid (ABS) and aniline (A) with different monomer ratios were synthesized. The P(AB‐ABS‐A) were grafted to the surface of multiwalled carbon nanotubes (MWCNTs). FTIR measurements prove the covalent binding to the carboxylic groups of the MWCNTs while conductivity tests show an increase in the conductivity of the nanohybrid in comparison to the polymers. The [MWCNT‐P(AB‐ABS‐A)] nanohybrids were used for the immobilization of redox active cytochrome c (cyt.c). The positively charged protein can electrostatically interact with the negatively charged nanohybrid. Cyclic voltammetry (CV) shows an increase in the protein loading on [MWCNT‐P(AB‐ABS‐A)] coupled to cysteamine modified gold electrodes in comparison to non‐grafted MWCNTs. A further increase in the sulfonation degree of P(AB‐ABS‐A) leads to an enhanced current output of the modified electrodes. The redox activity of the polymer decreases after the immobilization of the cyt.c on the nanohybrid. For the first time polymers covalently grafted to the surface of MWCNTs are used in a biosensor.     

Electrochemistry at chemically assembled single-wall carbon nanotube arrays

Single-wall carbon nanotubes (SWNTs) chemically assembled on gold substrates were employed as electrodes to investigate the charge transfer process between SWNTs and the underlying substrates. Cyclic voltammetry (CV) indicates that the assembled SWNTs allow electron communication between a gold electrode and the redox couple in solution, though the SWNTs are linked directly onto the insulating monolayer of 11-amino-n-undecanethiol (AUT) on the Au substrate. An electron transfer (ET) mechanism, which contains an electron tunneling process across the AUT monolayer, is proposed to explain the CV behavior of Au/AUT/SWNT electrodes. Electrochemical measurements show that the apparent electron tunneling resistance, which depends on the surface density of assembled SWNTs, has apparent effects similar to those of solution resistance on CV behavior . The theory of solution resistance is used to describe the apparent tunneling resistance. The experimental results of the dependence of ET parameter psi on the potential scan rate upsilon are in good agreement with the theoretical predictions. Kinetic studies of the chemical assembly of SWNTs by atomic force microscopic (AFM), electrochemical, and Raman spectroscopic methods reveal that two distinct assembly kinetics exist: a relatively fast step that is dominated by the surface reaction, and a successive slow step that is governed by bundle formation.