Modified Gold Substrate Research Articles

Synthesis, Electrochemical Properties and Self-Assembly of a Proton-Conducting Core-Shell Macromolecule

Let the protons flow: The synthesis of a core-shell macromolecule bearing phosphonic acids is presented. The rigid central core serves as a scaffold to stabilize the flexible polymer shells. Pronounced proton conductivity is obtained under humidified conditions. The self-assembly of such dendritic macromolecules by electrostatic interactions on a modified gold substrate is investigated and characterized.

Immobilization of galactose oxidase on self‐assembled monolayers of thiols on Au and Ag surfaces

Galactose oxidase (GalOD) was immobilized on self‐assembled monolayers of thiols on silver and gold surfaces using trans‐stilbene (4,4′‐diisothiocyanate)‐2,2′disulphonic acid (DIDS) as the bridging compound. DIDS is the symmetrical bifunctional reagent that reacted with the amine moiety of the thiol and with primary amino groups of enzyme. The Raman measurement revealed that onto cysteamine‐modified silver and gold electrodes, bands corresponding to the galactose oxidase (about 694, 1076, 1274 cm—1 on Au and 762, 1058, 1274 cm–1 on Ag ) appeared and clearly demonstrated its immobilization onto Au and Ag surfaces. Simultaneously, we have also observed changes in the ratio of trans–gauche conformers of adsorbed cysteamine molecules. Layers revealing high content of trans conformer are transformed into layers composed mainly of cysteamine molecule in gauche conformation after galactose oxidase adsorption. These observations deliver a strong support for enzyme immobilization on cysteamine‐modified gold and silver surfaces. The surface plasmon resonance experiment gave a surface coverage of ~8.4 × 107 g/cm2 for gold electrode modified cysteamine using DIDS chemistry and 1.1 × 107 g/cm2 for the cysteamine only modified gold substrate and demonstrated that galactose oxidase layers immobilized with DIDS coupling reagent are quite stable and cannot be easily removed from the surface by treatment with a buffer solution. The surface plasmon resonance results indicated that in this method, a multilayer of galactose oxidase have been immobilized. Our new method of covalent attachment of enzymes seems to be quite promising as a new way of manufacturing biosensors. Copyright © 2012 John Wiley & Sons, Ltd.

A Combined Normal Raman, Surface-Enhanced Raman Scattering (SERS), and Theoretical Calculation (DFT and PED) Study of Alpha-Tocopherol

In this work, the band assignment for the vibration mode of the functional groups contained in α-tocopherol (aT) molecules was analyzed entirely for the first time using their normal Raman spectra (NRS), density functional theory (DFT) calculations, and potential energy distributions (PED). The bands obtained through the theory calculation method were in accordance with experimentally observed values. The surface-enhanced Raman scattering (SERS) spectra of aT adsorbed on nanoparticles were studied in gold colloidal solutions and on modified gold substrates. Some crucial differences were observed in the SERS spectra of the aT molecules adsorbed on these two substrates, indicating that the behavior of aT adsorbed on gold colloids was different from that of aT on the modified gold substrate. The aT molecules probably lie flat on the surfaces of the gold nanoparticles through the ring in the gold colloidal solution, whereas they stand with a certain angle on the gold surfaces of the modified substrate. Furthermore, the concentration dependence of SERS spectra of aT is characterized by variation of the enhanced intensity of the bands, primarily suggesting that the adsorption behavior of the aT molecules adsorbed on the gold nanoparticles surface is changed with different concentrations. With the concentration around 7.5 × 10−2 mol/L, the sub-monolayer mode, and more aT molecules allowed to lie on the gold surface, then Raman signals are greatly enhanced in the SERS spectrum.

Biocompatible conductive architecture with surface-confined probe for non-invasive electrochemical cytosensing

The possibility of using electrochemical methods to investigate cell immobilization and natural physiological states provides great advances in life science research and public health protection. Herein, cytosensors with surface-confined ferrocene as signal indicator were developed to study the immobilization of human cervical carcinoma (HeLa) cells. With layer-by-layer (LBL) technique positively charged ferrocene functionalized poly(allylamine hydrochloride) (Fc-PAH) and negatively charged single-wall carbon nanotubes (SWNTs) were alternately assembled on 3-mercaptopropionic acid (MPA) modified gold substrates. The as-prepared cytosensors presented good biocompatibility and HeLa cells could keep viability for 72h on the materials according to the proliferation results. With differential pulse voltammetry (DPV) measurements the cytosensors exhibited high sensitivity to the detection of HeLa cells within a wide concentration range from 10 to 107cells/mL.

QCM study of the adsorption of polyelectrolyte covered mesoporous TiO2 nanocontainers on SAM modified Au surfaces

Mesoporous TiO2 nanocontainers (NCs) covered with polyelectrolyte multilayers were adsorbed on self-assembled monolayer (SAM) modified gold substrates at different values of pH and ionic strength. The adsorption process was followed in situ by means of a quartz crystal microbalance (QCM) and the morphology of the adsorbate was investigated by means of FE-SEM images taken of the substrates after each adsorption process. Deposition could be achieved if either the particles and the surface had opposite charge, or if the salt concentration was sufficiently high, reducing the repulsion between the spheres and the surface. In the latter case the adsorption kinetics could be explained in the context of the DLVO-theory. Using conditions of like charges, one has a means to control the speed of deposition by means of ionic strength. However, interparticle aggregation and cluster deposition on the surface were observed at high ionic strength. Such conditions have to be avoided to obtain a uniform deposition of separated nanocontainers on the surface.

Chemically bonded carbon nanotubes on modified gold substrate as novel unbreakable solid phase microextraction fiber

A new technique is introduced for preparation of an unbreakable fiber using gold wire as a substrate for solid phase microextraction (SPME). A gold wire is used as a solid support, onto which a first film is deposited that consists of a two-dimensional polymer obtained by hydrolysis of a self-assembled monolayer of 3-(trimethoxysilyl)-1-propanthiol. This first film is covered with a layer of 3-(triethoxysilyl)-propylamine. Next, a stationary phase of oxidized multi-walled carbon nanotubes was chemically bound to the surface. The synthetic strategy was verified by Fourier transform infrared spectroscopy and scanning electron microscopy. Thermal stability of new fiber was examined by thermogravimetric analysis. The applicability of the novel coating was verified by its employment as a SPME fiber for isolation of diazinon and fenthion, as model compounds. Parameters influencing the extraction process were optimized to result in limits of detection as low as 0.2 ng mL−1 for diazinon, and 0.3 ng mL−1 for fenthion using the time-scheduled selected ion monitoring mode. The method was successfully applied to real water, and the recoveries for spiked samples were 104% for diazinon and 97% for fenthion.

Peptide Separation through a CB[8]-Mediated Supramolecular Trap-and-Release Process

We demonstrate a supramolecular peptide separation approach by the selective immobilization of peptides bearing an N-terminal tryptophan onto a CB[8]-modified gold substrate, followed by electrochemical release. The CB[8]-stabilized heteroternary complexes were characterized by (1)H NMR, ESI-MS, UV/vis, and fluorescence spectroscopy and cyclic voltammetry. Micropatterned CB[8]-modified gold substrates were found to trap only the recognizable N-tryptophan-containing peptides from a peptide mixture that could be visualized as green peptide arrays under fluorescence microscopy. Subsequently, the bound peptides were released from the modified substrates by the controlled single-electron reduction of viologen. The fully reversible trap-and-release process was repeated for 13 cycles, and the cumulative release profile of the dye-peptide conjugate was monitored by fluorescence spectroscopy, indicating that no degradation occurred.

Imaging recognition events between human IgG and rat anti-human IgG by atomic force microscopy

Chemically immobilized rat anti-human immunoglobulin (IgG) monolayers on thiols modified gold substrates were fabricated using self-assembled monolayer (SAM) method. The antibody monolayers were imaged before and after free human IgG treated, whilst recognition events between antigen and antibody were monitored by contact mode atomic force microscopy (CM-AFM) and tapping mode AFM (TM-AFM), with topographic and/or phase images being recorded. The obtained images with different surface compositions show distinct nanostructures, indicating occurrence of recognition and binding events of antigen–antibody. The size of the observed surface structures of the antibody monolayer, when tip broaden effect had been taken into account, was very close to the actual size of the antibody molecule. Thus, these results suggest CM-AFM is capable of, and proven satisfactory in detecting protein–protein interactions (PPIs), providing the sample was prepared appropriately and the scanning parameters were set adequately. Moreover, phase imaging can serve as a real time contrast enhancement technique to TM-AFM in terms of highlighting edges and clearly observing fine features.

Surface plasmon resonance immunosensor for bacteria detection

This work describes an approach for the development of two bacteria biosensors based on surface plasmon resonance (SPR) technique. The first biosensor was based on functionalized gold substrate and the second one on immobilized gold nanoparticles. For the first biosensor, the gold substrate was functionalized with acid–thiol using the self-assembled monolayer technique, while the second one was functionalized with gold nanoparticles immobilized on modified gold substrate. A polyclonal anti- Escherichia coli antibody was immobilized for specific ( E. coli) and non-specific ( Lactobacillus) bacteria detection. Detection limit with a good reproducibility of 10 4 and 10 3 cfu mL −1 of E. coli bacteria has been obtained for the first biosensor and for the second one respectively. A refractive index variation below 5 × 10 −3 due to bacteria adsorption is able to be detected. The refractive index of the multilayer structure and of the E. coli bacteria layer was estimated with a modeling software.

Aryl diazonium salt surface chemistry and ATRP for the preparation of molecularly imprinted polymer grafts on gold substrates

AbstractQuercetin‐imprinted poly(vinyl pyridine‐co‐ethylene glycol dimethacrylate) cross‐linked grafts were prepared by atom transfer radical polymerization (ATRP) using aryl modified gold substrates as macroinitiators. The aryl layers were first attached to Au by electrochemical reduction of the diazonium salt BF$_{4}^{-}$, +N2‐C6H4‐CH2CH2‐OH. The resulting Au‐C6H4‐CH2CH2‐OH plates (Au‐OH) were further treated with 2‐bromopropionyl bromide (Br‐C(O)C(CH3)2‐Br) in order to attach ATRP initiator sites to Au‐OH, thus yielding Au‐C6H4‐CH2CH2‐OC(O)C(CH3)2‐Br (Au‐Br). 4‐Vinyl pyridine (4VP, functional monomer) and ethylene glycol dimethacrylate (EGDMA, cross‐linking monomer) were co‐polymerized in acetonitrile at room temperature in the presence of quercetin as a template molecule. Quercetin was then solvent‐extracted to obtain Au‐grafted molecularly imprinted polymer (Au‐MIP). The gold‐grafted nonimprinted polymer film (Au‐NIP) was prepared as a control substrate.The changes in the surface chemical composition, from the neat Au to Au‐MIP and Au‐NIP, were monitored by XPS and polarization modulation infrared reflection‐absorption spectroscopy (PM‐IRRAS). Cyclic voltammetry (CV) was used to assess the specificity and selectivity of Au‐MIP to detect quercetin. The detection limit was found to be better than 10−6 mol/l with CV. Using square wave stripping voltammetry (SWSV), the detection limit was found to be as low as 10−9 mol/l. Copyright © 2010 John Wiley & Sons, Ltd.

Layer-by-layer assembly of polysaccharide-based nanostructured surfaces containing polyelectrolyte complex nanoparticles

Nanoscale chemical and topographical features have been demonstrated to influence a variety of significant responses of mammalian cells to biomaterials surfaces. Thus, an important goal for biomaterials scientists is the ability to engineer the nanoscale surface features of biologically active materials. The goal of the current work is to demonstrate that polyelectrolyte complex nanoparticles (PCNs) in polyelectrolyte multilayers (PEMs) can be combined to create surfaces with controlled nanoscale surface topography and nanoscale presentation of surface chemistry. The polysaccharides used in this work are the biomedically relevant chitosan, heparin, and hyaluronan. Nanostructured surface coatings were characterized on both modified gold substrates and tissue-culture polystyrene surfaces. PCNs were adsorbed to oppositely charged PEMs, and were also embedded within PEMs. The construction of the surface coatings was characterized by quartz crystal microbalance with dissipation (QCM-D). The surface morphology was characterized by scanning electron microscopy (SEM) and atomic force microscopy (AFM). The chemistry of the coatings was confirmed by both X-ray photoelectron spectroscopy (XPS) and polarization modulation infra-red reflection absorption spectroscopy (PM-IRRAS). Morphologically, we found that PCNs were colloidally stable and homogeneously distributed when adsorbed on or in the PEMs. Chemical analysis confirms that the PCNs adsorbed to PEMs significantly altered the surface chemistry, indicating significant surface coverage. Furthermore, the position of the PCNs normal to the surface can be adjusted by adding PEMs on top of adsorbed PCNs. Thus, PCNs can be used to introduce discrete nanoscale surface topographical features and varying surface chemistry into PEM surface coatings in a controlled way.

Scanning electrochemical microscopy assay of DNA based on hairpin probe and enzymatic amplification biosensor

A novel scheme for scanning electrochemical microscopy (SECM) assay of DNA based on hairpin probe and enzymatic amplification biosensor was described. In this method, streptavidin–horseradish peroxidase (HRP) was captured by double-stranded DNA (ds-DNA) modified gold substrate via biotin–streptavidin interaction after hybridization of target DNA to the immobilized hairpin probe functioned with a biotin at its 3′ end. In the presence of H 2O 2, hydroquinone (H 2Q) was oxidized to benzoquinone (BQ) at the modified substrate surface through the HRP catalytic reaction, and the generated BQ corresponding to the amount of target DNA was reduced in solution by a SECM tip. The resulting reduction current allowed concentration detection of target DNA and SECM imaging of hybridization between the target DNA and the immobilized hairpin probe. The detection limit of this method was as low as 17 pM for complementary target DNA and it had good selectivity to discriminate between the complementary sequence and one containing base mismatches.

Synthesis and characterization of novel glycosurfaces by ATRP

The direct synthesis of well defined sugar methacrylate-based homopolymer brushes with high grafting densities based on D-gluconamidoethyl methacrylate (GAMA) and 2-lactobionamidoethyl methacrylate (LAMA) from functionalized gold substrates was carried out using surface-initiated atom transfer radical polymerization (ATRP). A good control of the polymerization leading to an increase in the dry film thickness with reaction time was achieved in mixed methanol/water solvent media. However, grafted glycopolymer films of low thicknesses, which did not increase further with longer polymerization times were synthesized in water, suggesting the premature termination of the polymerization in the aqueous solvent. Attenuated total reflectance (ATR)-FTIR spectroscopy confirmed the successful grafting of the glycopolymer brushes on the modified gold substrates, while atomic force microscopy (AFM) verified that the anchored film covered the substrate surface completely and homogeneously. The surface roughness found by AFM was below 1 nm suggesting the preparation of very smooth glycopolymer films. The grafting of the glycopolymer chains onto the gold substrates afforded an increase in the surface hydrophilicity as confirmed by contact angle measurements. The synthesized glycopolymer films exhibited strong binding interactions with specific lectins via the “glycocluster” effect.

Poly(ethylene glycol) Gradient for Biochip Development

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.

Characterization of Pore Formation by Streptolysin O on Supported Lipid Membranes by Impedance Spectroscopy and Surface Plasmon Resonance Spectroscopy

We report the study of the interactions of bacterial toxin streptolysin O (SLO) and cholesterol-containing membranes using electrochemical impedance and surface plasmon resonance (SPR) spectroscopy at low hemolytic units on a novel supported membrane interface. The detailed understanding of the process will aid significantly the construction of nanoscale transport channels for biosensing applications. Cholesterol-containing egg PC vesicles, pristine and incubated with SLO toxin, were fused onto a hexyl thioctate (HT)-modified gold substrate. The charge-transfer resistance of the resulting lipid membrane, which is related to the formation of the transmembrane pores, is measured with the aid of an electroactive probe. Impedance spectra were collected over a range of 0.1-100 kHz, and the obtained complex resistance was fit to an equivalent circuit. The charge-transfer resistance decreases for increasing SLO concentration, following a first-order exponential decay. The two-part membrane interface was further characterized with SPR spectroscopy. For the hexyl thioctate support layer, an equivalent monolayer thickness of 1.3 nm was determined. This value suggests a loosely packed structure of the monolayer on gold, presenting an ideal platform for permeability studies. A comparative study on the fusion behavior of vesicles with and without SLO induced pores revealed no substantial difference for the two systems, indicating that the pore formation has no adverse effect on the integrity of the vesicles. The resulting lipid membrane thickness from pre-perforated lipids was found to be 3.2 nm, suggesting that one leaflet is knocked off during the fusion process and a hybrid membrane is formed. A slightly higher thickness value of 3.4 nm was obtained for membranes from non-perforated vesicles. Deposition of lipids and subsequent incubation with SLO, as monitored by SPR, shows that the HT surface chemistry allows partial insertion of the toxin into the membrane, indicating unique properties as compared to the previously explored long-chain alkylthiols.

Electrochemistry and photoelectrochemistry of photosystem I adsorbed on hydroxyl-terminated monolayers

Direct electrochemistry studies on Photosystem I (PSI) were performed using cyclic voltammetry and square wave voltammetry. PSI centers stabilized in aqueous solution by Triton X-100 surfactant were adsorbed on hydroxyl-terminated hexanethiol modified gold electrodes. We have identified the electron donor, P700, and the electron acceptor sites, F A / F B , based on the previously reported preferred orientation for P700 on hydroxyl-terminated self-assembled monolayers. The reported potential values ( E P700 = +0.51 V vs. NHE; E FA/FB = −0.36 V vs. NHE) correlate very well with the established literature for P700, while the weaker signal for F A / F B lies within previous literature values. We were able to identify both redox centers on the same voltammogram. The P700 center clearly shows reversible electrochemical behavior. The expected F A / F B reduction is small in comparison, reflecting the dominant orientation of PSI with the F A / F B centers farther away and the P700 center nearer the electrode surface. As a molecular diode, PSI does not permit reverse direction conductivity to the F B so the small F A / F B peaks reflect other orientations besides the predominant one. PSI adsorbed on a hydroxyl-terminated hexanethiol modified gold substrate displays a photoenhanced reduction current for the P700 + center in the presence of light and an electron acceptor, methyl viologen. This photoelectrochemical response demonstrates protein functionality after adsorption.

Dendrimer-functionalized self-assembled monolayers as a surface plasmon resonance sensor surface.

We report here a multistep route for the immobilization of DNA and proteins on chemically modified gold substrates using fourth-generation NH(2)-terminated poly(amidoamine) dendrimers supported by an underlying amino undecanethiol (AUT) self-assembled monolayer (SAM). Bioactive ultrathin organic films were prepared via layer-by-layer self-assembly methods and characterized by fluorescence microscopy, variable angle spectroscopic ellipsometry, atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS), and attenuated total internal reflection Fourier transform infrared spectroscopy (ATR-FTIR). The thickness of the AUT SAM base layer on the gold substrates was determined to be 1.3 nm from ellipsometry. Fluorescence microscopy and AFM measurements, in combination with analyses of the XPS/ATR-FTIR spectra, confirmed the presence of the dendrimer/biopolymer molecules on the multilayer sensor surfaces. Model proteins, including streptavidin and rabbit immunoglobulin proteins, were covalently attached to the dendrimer layer using linear cross-linking reagents. Through surface plasmon resonance measurements, we found that sensor surfaces containing a dendrimer layer displayed an increased protein immobilization capacity, compared to AUT SAM sensor surfaces without dendrimer molecules. Other SPR studies also revealed that the dendrimer-based surfaces are useful for the sensitive and specific detection of DNA-DNA interactions. Significantly, the multicomponent films displayed a high level of stability during repeated regeneration and hybridization cycles, and the kinetics of the DNA-DNA hybridization process did not appear to be influenced by surface mass transport limiting effects.

Synthesis and Self-Assembly of Globotriose Derivatives: A Model System for Studies of Carbohydrate−Protein Interactions

Self-assembled monolayers (SAMs) on gold exposing α-d-Galp-(1→4)-β-d-Galp-(1→4)-β-d-Glcp (globotriose) are described. A synthetic pathway for the preparation of the bioactive carbohydrate globotriose coupled directly to bis(16-hydroxyhexadecanyl) disulfide ((HOC16H32S)2), as well as via tetra- or di(ethylene glycol) spacers, was developed. The SAMs were characterized by ellipsometry, contact angle goniometry, infrared reflection−absorption spectroscopy, and by their interactions with monoclonal antibodies. The ellipsometry measurements of mixed SAMs revealed thicknesses between 22 and 40 A, depending on the ratio between carbohydrate and non-carbohydrate disulfides in the preparation solution. When the solution contained 10% or more of the carbohydrate adsorbate, the modified gold substrates displayed total wetting. Infrared reflection−absorption spectroscopy conferred well-ordered SAMs with a high degree of crystallinity. Furthermore, two monoclonal antibodies (IgM, MAHI 419 and IgG, MAHI 5) showed diffe...

Immobilizing Shortened Single-Walled Carbon Nanotubes (SWNTs) on Gold Using a Surface Condensation Method

We propose a surface condensation method for assembling single-walled carbon nanotubes (SWNTs) on gold. The as-prepared long and randomly tangled SWNTs were cut into short pipes by chemical oxidation, allowing the nanotubes to be terminated by carboxyl functionalities. A surface condensation reaction was then performed by immersing an amino self-assembled monolayer (SAM)-modified gold substrate into the dimethylformamide suspension of carboxylic nanotubes with the aid of dicyclohexylcarbodiimide condensation agent. Raman spectroscopy and atomic force microscopy (AFM) results show that a highly aligned assembly of SWNTs has been formed on gold, with the nanotubes standing on the surface stable enough for a long ultrasonication. In combination with the microcontact printing (μCP) technique, we have fabricated patterned nanotube assemblies using this surface condensation method. Moreover, we found that the “giant” carbon nanotubes tend to form bundles on an amino-terminating surface, likely following a nucleation-growth model.

Ultrathin Polymer Films on Gold Surfaces through Activation of Si–H Bonds

Several poly(siloxane)s and a poly(silazane) were bound to gold surfaces by activation of Si–H bonds with cis-[PtCl2(PhCHCH2)2]. Under the reaction conditions used here, adsorption of the poly(siloxane) is observed exclusively in presence of the platinum compound. The resulting ultrathin layers were studied with IR spectroscopy at grazing incidence reflection, X-ray photoelectron spectroscopy, contact angle measurements, and ellipsometry. The layer thicknesses (2.9–5.2 nm) are in the typical range of polymer monolayers adsorbed on gold. The wetting properties of the modified gold substrates correspond to those of the pure polymers, indicating that the gold is covered to a high extent or completely with polymer. The highest contact angles of water (115°) were observed with Si–H-terminated poly(dimethylsiloxane) and are among the highest reported for an ultrathin layer on gold. We assume that the activator cleaves Si–H bonds. The polymers might be bound to the surface by Au–Si bonds or via Pt–Si species.