Modified Gold Substrate Research Articles

Growth of Lactic Acid Bacteria on Gold-Influence of Surface Roughness and Chemical Composition.

The main focus of this work was to establish a correlation between surface topography and chemistry and surface colonization by lactic acid bacteria. For this reason, we chose gold substrates with different surface architectures (i.e., smooth and nanorough) that were characterized by atomic force microscopy (AFM), electron scanning microscopy (SEM), and X-ray diffractometry (XRD). Moreover, to enhance biocompatibility, we modified gold substrates with polymeric monolayers, namely cationic dextran derivatives with different molar masses. The presence of those layers was confirmed by AFM, infrared spectroscopy (IR), and X-ray photoelectron spectroscopy (XPS). In order to determine the adhesion abilities of non-modified and modified gold surfaces, we tested three lactic acid bacteria (LAB) strains (i.e., Lactobacillus rhamnosus GG, Lactobacillus acidophilus, and Lactobacillus plantarum 299v). We have shown that surface roughness influences the surface colonization of bacteria, and the most significant impact on the growth was observed for the Lactobacillus rhamnosus GG strain. What is more, covering the gold surface with a molecular polymeric film by using the layer-by-layer (LbL) method allows additional changes in the bacterial growth, independently on the used strain. The well-being of the bacteria cells on tested surfaces was confirmed by using selective staining and fluorescence microscopy. Finally, we have determined the bacterial metabolic activity by measuring the amount of produced lactic acid regarding the growth conditions. The obtained results proved that the adhesion of bacteria to the metallic surface depends on the chemistry and topography of the surface, as well as the specific bacteria strain.

Indirect surface-enhanced Raman scattering assay of insulin-like growth factor 2 receptor protein by combining the aptamer modified gold substrate and silver nanoprobes.

An indirect aptamer-based SERS assay for insulin-like growth factor 2 receptor (IGF-IIR) protein was developed. The gold substrate and silver nanoparticles (AgNPs) were employed simultaneously to achieve double enhancement for SERS signals. Firstly, the five commercial SERS substrates including Enspectr, Ocean-Au, Ocean-AG, Ocean-SP and Q-SERS substrates were evaluated using 4-mercaptobenzoic acid (4-MBA). The Q-SERS substrate was selected based on low relative standard deviation (RSD, 8.6%) and high enhancement factor (EF, 8.7*105), using a 785nm laser. The aptamer for IGF-IIR protein was designed to include two sequences: one grafted on gold substrate to specifically capture the IGF-IIR protein and a second one forming a 3' sticky bridge to capture SERS nanotags. The SERS nanotag was composed by AgNPs (20nm), 4-MBA and DNA probes that can hybridize with the aptamer. Due to the steric-hindrance effect, when the aptamer doesn't combine with IGF-IIR protein, it only can capture the SERS nanotags. Therefore, there was a negative correlation between the concentration of IGF-IIR protein and the intensity of 4-MBA at 1076cm-1. The detection limit reached to 141.2 fM and linear range was from 10 pM to 1μM. The SERS aptasensor also exhibits a high reproducibility with an average RSD of 4.5%. The interference test was conducted with other four proteins to verify the accuracy of measuring. The study provides an approach to quantitative determination of proteins based on specific recognition and nucleic acid hybridization of aptamers, to establish sandwich structure for SERS enhancement. Graphical abstractSchematic representation of surface-enhanced Raman scattering (SERS) assay on insulin-like growth factor 2 receptor (IGF-IIR) protein by combining the aptamer modified gold substrate and 4-mercaptobenzoic acid (4-MBA) and DNA probe modified silver nanoparticles.

Influence of intermolecular H-bonding on the acid-base interfacial properties of -COOH and ferrocene terminated SAM

We have explored the intermolecular interactions within a two-component self-assembled monolayer, a model system to develop our understanding of interfacial chemistry in molecular level. The redox signal of 11-ferrocenyl-1-undecanethiol (FcC11) reversibly responded to the formation of intermolecular H-bonding: The intermolecular H-bonding between the terminal carboxyl groups of MSA promoted the formation of MSA assemblies, which increased the influence of MSA on the neighboring molecules (FcC11). Meanwhile, the disordered FcC11 appeared within the locally ordered FcC11 patches, resulting in the presence of a new couple of redox peaks in the cyclic voltammograms. The surface coverage that corresponding to the H-bonding affected FcC11 increased exponentially from 0 to 3.49 × 10−10 mol cm−2 as a function of the time of MSA assembly. The pK1/2 value (the pH when half of the –COOH groups are ionized) that determined for the FcC11/MSA modified gold substrate was obviously higher than the alkanoic acids in aqueous solution. The results show that the intermolecular H-bonding between MSA molecules plays a key role in controlling the molecular assembly within the two-component self-assembled monolayer, which provides an approach for regulating the structural changes of the model membrane in molecular level.

The Effect of Cholesterol on the Dielectric Structure of Lipid Bilayers.

Cholesterol plays an important role in regulating the properties of phospholipid bilayers and many mechanisms have been proposed to explain why cholesterol is so ubiquitous within biological membranes of animals. Here we present the results of studies on the effect of cholesterol on the electrical/dielectric properties of lipid membranes tethered to a solid substrate. These tethered bilayer lipid membranes tBLM were formed on a commercially available chemically modified gold substrate. These lipid bilayers are very robust. Very high-resolution electrical impedance spectroscopy (EIS) was used to determine the dielectric structure of the lipid bilayers and associated interfaces. The EIS data allowed the dielectric substructure of the lipid bilayers to be determined. The results showed that when cholesterol was present in the tethered membranes at a concentration of 10% (mol/mol); the thickness of the tBLMs increased and the membrane conductance decreased. However, when cholesterol was present in the tethered membrane at more than 30% (mol/mol) the effect of cholesterol was dramatically different; the membranes then became thinner and possessed a much larger electrical conductance. The EIS allowed a distinction to be made between a hydrophobic region in the center of the bilayer and another hydrophobic region further out towards the polar head region, in addition to the polar head region itself. Cholesterol was found to have the largest effect on the inner, hydrophobic region, although the outer hydrophobic region was also affected.

Supramolecular Cyclodextrin Supplements to Improve the Tissue Adhesion Strength of Gelatin Bioglues.

A practical strategy to strengthen the inherent tissue adhesiveness of bioglues was investigated without compromising their chemical structure. A simple blending of α-cyclodextrins (α-CDs) in gelatin glues substantially improved the adhesiveness of the glues upon contact with porcine skin. Interestingly, the adhesiveness was even further enhanced when the glues were supplemented with γ-CDs through the formation of multivalent supramolecular networks inside of the glue. In contrast, the effect of β-CDs was rather limited because their water solubility is relatively low. A model study using modified gold substrates demonstrated that CDs also interacted with biomolecules naturally present in skin, thereby improving the adhesiveness. Altogether, we suggest that the supramolecular networking of CDs, both inside the glue and at the glue-skin interface, could reinforce tissue adhesiveness of bioglues, which allows them to be of great relevance to a primary treatment modality for larger skin incisions.

The Effect of Benzyl Alcohol on the Dielectric Structure of Lipid Bilayers.

Molecularly tethered lipid bilayer membranes were constructed on a commercially available chemically modified gold substrate. This is a new and promising product that has allowed the construction of very robust lipid bilayers. Very high resolution electrical impedance spectroscopy (EIS) was used to determine the dielectric structure of the lipid bilayers and associated interfaces. The EIS data were modelled in terms of the dielectric substructure using purpose developed software. The hydrophobic region, where the lipid tails are located, was revealed by the EIS in the frequency range of (1-100) Hz and its thickness was calculated from the capacitance of this region and found to be approximately 3-4nm. The hydrophilic region, where the polar heads are located, was revealed at higher frequencies and its thickness was estimated to be approximately 1-2nm. The effect of the local anaesthetic benzyl alcohol (BZA) on the tethered lipid bilayers was investigated. The effect of BZA on the membrane capacitance and conductance allowed the changes in the thickness of the polar head and hydrophobic tails regions to be determined. It was found that the addition of BZA caused a significant increase in the capacitance (corresponding to a decrease in the thickness) of the hydrophobic region and an increase in the membrane electrical conductance. The EIS allowed a distinction between a hydrophobic region in the centre of the bilayer and an outer hydrophobic region. Benzyl alcohol was found to have the largest effect on the outer, hydrophobic region, although the inner hydrophobic region was also consistently affected.

Voltammetric behavior of selenocystine at modified gold substrates

The voltammetric response of seleno-l-cystine has been studied at gold substrates under physiological conditions. The reactivity of diselenides is utilized to generate a modified electrode surface, which in turn allows for electroanalysis of solution-based selenium species. Stable and reproducible voltammetry is observed for selenocystine reduction at pH 7 on selenium-modified gold substrates (Emid=−486mV vs. Ag/AgCl) and is consistent with a diffusionally controlled process. Modification of the gold surface is readily achieved via electrochemical cycling in the presence of a diselenide source at conventional scan rates. These studies afford voltammetric characterization of the selenocystine/selenocysteine redox couple under physiologically relevant conditions and highlight the potential utility of selenium-modified substrates for electroanalysis of chalcogen-containing species.

Characterization of a Self-Assembled Monolayer of 1-Thio-β-D-Glucose with Electrochemical Surface Enhanced Raman Spectroscopy Using a Nanoparticle Modified Gold Electrode.

Preparation of a nanoparticle modified gold substrate designed for characterization of hydrophilic self-assembled monolayers (SAMs) of 1-thio-β-D-glucose (TG) with electrochemical surface-enhanced Raman spectroscopy (EC-SERS) is presented. Citrate stabilized gold nanoparticles were deposited on a polycrystalline gold electrode and subjected to an electrochemical desorption procedure to completely remove all traces of adsorbed citrate. Complete desorption of citrate was confirmed by recording cyclic voltammetry curves and SERS spectra. The citrate-free nanoparticle modified gold electrode was then incubated in a 1 mg mL(-1) aqueous solution of TG for 16 h prior to being characterized by EC-SERS. The SERS spectra confirmed that at potentials more negative than -0.10 V vs SCE thioglucose forms a monolayer in which the majority of the molecules preserve their lactol ring structure and only a small fraction of molecules appear to be oxidized. At potentials more positive than -0.10 V, the oxidation of TG molecules becomes prominent, and at potentials more positive than 0.20 V vs SCE, the monolayer of TG consists chiefly of oxidized product. The SERS spectra collected in the double layer region suggest the SAM of TG is well hydrated and hence can be used for hydrophilic modifications of a gold surface.

Luminol chemiluminescence biosensor for glycated hemoglobin (HbA1c) in human blood samples.

Luminol chemiluminescence (CL) biosensor based on boronic acid modified gold substrate has been developed for the determination of glycated hemoglobin (HbA1c) in human blood samples. In order to selectively capture HbA1c in sample, carboxy-EG6-undecanethiol was self-assembled on a gold thin-film substrate, followed by covalent coupling of 3-aminophenyl boronic acid (3-APBA). The captured HbA1c containing four iron heme groups plays as a catalyst for luminol CL reaction in the presence of hydrogen peroxide, and thus the luminol CL response is linearly proportional to the amount of HbA1c captured on the biosensor surface. The present biosensor showed linear dynamic range of HbA1c from 2.5% to 17.0%, which well covers the clinically important concentration range. In addition, the present biosensor exhibited negligible response to interfering species such as hemoglobin, fructose, and sorbitol. The present HbA1c biosensor was applied to the determination of HbA1c in human blood samples and the results were well agreed with that obtained with a conventional method.

On the molecular interaction between albumin and ibuprofen: An AFM and QCM-D study

The adsorption of proteins on surfaces often results in a change of their structural behavior and consequently, a loss of bioactivity. One experimental method to study interactions on a molecular level is single molecular force spectroscopy that permits to measure forces down to the pico-newton range.In this work, the binding force between human serum albumin (HSA), covalently immobilized on glutaraldehyde modified gold substrates, and ibuprofen sodium salt was studied by means of single molecular force spectroscopy. First of all, a protocol was established to functionalize atomic force microscopy (AFM) tips with ibuprofen. The immobilization protocol was additionally tested by quartz crystal microbalance with dissipation (QCM-D) and contact angle measurements. AFM was used to characterize the adsorption of HSA on gold substrates, which lead to a packed monolayer of thickness slightly lower than the reported value in solution.Finally, single molecule spectroscopy results were used to characterize the binding force between albumin and ibuprofen and calculate the distance of the transition state (0.6nm) and the dissociation rate constant (0.055s−1). The results might indicate that part of the adsorbed protein still preserves its functionality upon adsorption.

Chirality-assisted ring-like aggregation of aβ(1-40) at liquid-solid interfaces: a stereoselective two-step assembly process.

Molecular chirality is introduced at liquid-solid interfaces. A ring-like aggregation of amyloid Aβ(1-40) on N-isobutyryl-L-cysteine (L-NIBC)-modified gold substrate occurs at low Aβ(1-40) concentration, while D-NIBC modification only results in rod-like aggregation. Utilizing atomic force microscope controlled tip-enhanced Raman scattering, we directly observe the secondary structure information for Aβ(1-40) assembly in situ at the nanoscale. D- or L-NIBC on the surface can guide parallel or nonparallel alignment of β-hairpins through a two-step process based on electrostatic-interaction-enhanced adsorption and subsequent stereoselective recognition. Possible electrostatic interaction sites (R5 and K16) and a chiral recognition site (H14) of Aβ(1-40) are proposed, which may provide insight into the understanding of this effect.

Chirality‐Assisted Ring‐Like Aggregation of Aβ(1–40) at Liquid–Solid Interfaces: A Stereoselective Two‐Step Assembly Process

AbstractMolecular chirality is introduced at liquid–solid interfaces. A ring‐like aggregation of amyloid Aβ(1–40) on N‐isobutyryl‐L‐cysteine (L‐NIBC)‐modified gold substrate occurs at low Aβ(1–40) concentration, while D‐NIBC modification only results in rod‐like aggregation. Utilizing atomic force microscope controlled tip‐enhanced Raman scattering, we directly observe the secondary structure information for Aβ(1–40) assembly in situ at the nanoscale. D‐ or L‐NIBC on the surface can guide parallel or nonparallel alignment of β‐hairpins through a two‐step process based on electrostatic‐interaction‐enhanced adsorption and subsequent stereoselective recognition. Possible electrostatic interaction sites (R5 and K16) and a chiral recognition site (H14) of Aβ(1–40) are proposed, which may provide insight into the understanding of this effect.

Effect of surface potential on extracellular matrix protein adsorption.

Extracellular matrix (ECM) proteins, such as fibronectin, laminin, and collagen IV, play important roles in many cellular behaviors, including cell adhesion and spreading. Understanding their adsorption behavior on surfaces with different natures is helpful for studying the cellular responses to environments. By tailoring the chemical composition in binary acidic (anionic) and basic (cationic) functionalized self-assembled monolayer (SAM)-modified gold substrates, variable surface potentials can be generated. To examine how surface potential affects the interaction between ECM proteins and substrates, a quartz crystal microbalance with dissipation detection (QCM-D) was used. To study the interaction under physiological conditions, the ionic strength and pH were controlled using phosphate-buffered saline at 37 °C, and the ζ potentials of the SAM-modified Au and protein were determined using an electrokinetic analyzer and phase analysis light scattering, respectively. During adsorption processes, the shifts in resonant frequency (f) and energy dissipation (D) were acquired simultaneously, and the weight change was calculated using the Kelvin-Voigt model. The results reveal that slightly charged protein can be adsorbed on a highly charged SAM, even where both surfaces are negatively charged. This behavior is attributed to the highly charged SAM, which polarizes the protein microscopically, and the Debye interaction, as well as other short-range interactions such as steric force, hydrogen bonding, direct bonding, charged domains within the protein structure, etc., that allow adsorption, although the macroscopic electrostatic interaction discourages adsorption. For surfaces with a moderate potential, proteins are not significantly polarized by the surface, and the interaction can be predicted through simple electrostatic attraction. Furthermore, surface-induced self-assembly of protein molecules also affects the adsorbed structures and kinetics. The adsorbed layer properties, such as rigidity and packing behaviors, were further investigated using the D-f plot and phase detection microscopy (PDM) imaging.

Gold‐Nanoparticle‐Assisted Self‐Assembly of Chemical Gradients with Tunable Sub‐50 nm Molecular Domains

A simple and efficient principle for nanopatterning with wide applicability in the sub‐50 nanometer regime is chemisorption of nanoparticles; at homogeneous substrates, particles carrying surface charge may spontaneously self‐organize due to the electrostatic repulsion between adjacent particles. Guided by this principle, a method is presented to design, self‐assemble, and chemically functionalize gradient nanopatterns where the size of molecular domains can be tuned to match the level corresponding to single protein binding events. To modulate the binding of negatively charged gold nanoparticles both locally (<100 nm) and globally (>100 μm) onto a single modified gold substrate, ion diffusion is used to achieve spatial control of the particles’ mutual electrostatic interactions. By subsequent tailoring of different molecules to surface‐immobilized particles and the void areas surrounding them, nanopatterns are obtained with variable chemical domains along the gradient surface. Fimbriated Escherichia coli bacteria are bound to gradient nanopatterns with similar molecular composition and macroscopic contact angle, but different sizes of nanoscopic presentation of adhesive (hydrophobic) and repellent poly(ethylene) glycol (PEG) domains. It is shown that small hydrophobic domains, similar in size to the diameter of the bacterial fimbriae, supported firmly attached bacteria resembling catch‐bond binding, whereas a high number of loosely adhered bacteria are observed on larger hydrophobic domains.

Excitation energy migration in yellow fluorescent protein (citrine) layers adsorbed on modified gold surfaces

The nature of functional proteins adsorbed on solid surfaces is interesting from the perspective of developing of bioelectronics and biomaterials. Here we present evidence that citrine (one of yellow fluorescent protein variants) adsorbed on modified gold surfaces would not undergo denaturation and energy transfer among the adsorbed citrine molecules would occur. Gold substrates were chemically modified with 3-mercaptopropionic acid and tert-butyl mercaptan for the preparation of hydrophilic and hydrophobic surfaces, respectively. A pure solution of citrine was dropped and dried on the modified gold substrates and their surface morphology was studied with scanning tunnelling microscopy (STM). The obtained STM images showed multilayers of citrine adsorbed on the modified surfaces. On hydrophobic surfaces, citrine was adsorbed more randomly, formed various non-uniform aggregates, while on hydrophilic surfaces, citrine appeared more aligned and isolated uniform protein clusters were observed. Fluorescence lifetime and anisotropy decay of these dried citrine layers were also measured using the time correlated single photon counting method. Fluorescence anisotropy of citrine on the hydrophobic surface decayed faster than citrine on the hydrophilic surface. From these results we concluded that fluorescence energy migration occurred faster among citrine molecules which were randomly adsorbed on the hydrophobic surface to compare with the hydrophilic surface.

Palladium nanoparticles on electrochemically reduced chemically modified graphene oxide for non-enzymatic bimolecular sensing

This paper reports on the design, fabrication, and characterization of palladium nanoparticles (PdNPs) on electrochemically reduced chemically modified graphene oxide (ERCGO) modified gold substrate for enzyme-free bimolecular detection. The ERCGO-modified electrode was prepared electrochemically, which is a fast and controllable process due to the high cathodic reduction of oxygen functional groups of chemically modified graphene oxide on the surface of a gold substrate electrode in sodium hydroxide (NaOH) solution. Then, PdNPs were deposited electrochemically, which were dispersed homogeneously on the surface of ERCGO. The fabricated hybrid electrode exhibited good performance, along with low working potential, high sensitivity, low detection limit, and long-term stability, which are attributed to the high surface area of ERCGO that enabled the deposition of PdNPs. This electrode offers great promise for a new class of nanostructured electrodes, for non-enzymatic biosensor applications.

Highly sensitive and selective detection of cancer cell with a label-free electrochemical cytosensor

Electrochemical methods have attracted considerable attention for developing cytosensing system since they can decrease the cost and time requirement for cell detection with simple instrumentation. Herein, a label-free electrochemical cytosensor with surface-confined ferrocene as signal indicator was developed for highly sensitive and selective detection of cancer cell. With layer-by-layer (LBL) self-assembly technique, positively charged poly(ethylene imine) functionalized with ferrocene (Fc-PEI) and negatively charged single-wall carbon nanotubes (SWNTs) were alternately assembled on 3-mercaptopropionic acid (MPA) modified gold substrate. Folic acid (FA) was covalently bonded onto SWNTs surface to specifically recognize cancer cells according to the high affinity of FA for folate receptor (FR) on cellular surface. The developed cytosensor presented high sensitivity and selectivity for the detection of human cervical carcinoma (HeLa) cell. By using fast-response differential pulse voltammetry (DPV) method, a wide detection range from 10 to 106cells/mL with a detection limit as low as 10cells/mL was reached even in the presence of a large amount of non-cancerous cells.

Adsorption behavior of plasmid DNA on binary self-assembled monolayers modified gold substrates

Gold is known to have good biocompatibility because of its inert activity and the surface property can be easily tailored with self-assembled monolayers (SAMs). In previous works, gold surfaces were tailored with homogeneously mixed amine and carboxylic acid functional groups to generate surfaces with a series of isoelectronic points (IEPs). In other words, by tailoring the chemical composition in binary SAMs, different surface potentials can be obtained under controlled pH environments. To understand how the surface potentials affect the interaction at the interface, a binary-SAMs-modified Au electrode on a quartz crystal microbalance with dissipation detection (QCM-D) was used owing to the high weight sensitivity of QCM-D. In QCM-D, the frequency shift and the energy dissipation are monitored simultaneously to determine the adsorption behaviors of the plasmid DNA to surfaces of various potentials in Tris-buffered NaCl solutions of different pH. The results revealed that the plasmid DNA can be adsorbed on the SAM-modified surfaces electrostatically; thus, in general, the amount of adsorbed plasmid DNA decreased with increasing environmental pH and the decreasing ratio of the amine functional groups on the surfaces owing to weaker positive potentials on the surface. For the high amine-containing surfaces, due to the strong electrostatic attraction, denser films were observed, and thus, the apparent thickness decreased slightly. The negatively charged carboxylic acid surfaces can still adsorb the negatively charged plasmid DNA at some conditions. In other words, the electrostatic model cannot explain the adsorption behavior completely, and the induced dipole (Debye) interaction between the charged and polarizable molecules needs to be considered as well.

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.

Detection to E. Coli O157 by Surface Plasmon Resonance Immunosensor

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 silver 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 silver nanoparticles immobilized on modified gold substrate. A polyclonal anti-Escherichia coli antibody was immobilized for specific (E. coli) bacteria detection. Detection limit with a good reproducibility of 104 and 103 cfu•mL−1 of E. coli bacteria has been obtained for the first biosensor and for the second one respectively.