Mixed Self-assembled Monolayers Research Articles

Reversible Biofunctionalization of Surfaces with a Switchable Mutant of Avidin

Label-free biosensors detect binding of prey molecules (″analytes″) to immobile bait molecules on the sensing surface. Numerous methods are available for immobilization of bait molecules. A convenient option is binding of biotinylated bait molecules to streptavidin-functionalized surfaces, or to biotinylated surfaces via biotin-avidin-biotin bridges. The goal of this study was to find a rapid method for reversible immobilization of biotinylated bait molecules on biotinylated sensor chips. The task was to establish a biotin-avidin-biotin bridge which was easily cleaved when desired, yet perfectly stable under a wide range of measurement conditions. The problem was solved with the avidin mutant M96H which contains extra histidine residues at the subunit-subunit interfaces. This mutant was bound to a mixed self-assembled monolayer (SAM) containing biotin residues on 20% of the oligo(ethylene glycol)-terminated SAM components. Various biotinylated bait molecules were bound on top of the immobilized avidin mutant. The biotin-avidin-biotin bridge was stable at pH ≥3, and it was insensitive to sodium dodecyl sulfate (SDS) at neutral pH. Only the combination of citric acid (2.5%, pH 2) and SDS (0.25%) caused instantaneous cleavage of the biotin-avidin-biotin bridge. As a consequence, the biotinylated bait molecules could be immobilized and removed as often as desired, the only limit being the time span for reproducible chip function when kept in buffer (2-3 weeks at 25 °C). As expected, the high isolectric pH (pI) of the avidin mutant caused nonspecific adsorption of proteins. This problem was solved by acetylation of avidin (to pI < 5), or by optimization of SAM formation and passivation with biotin-BSA and BSA.

Steric Hindrance of Photoswitching in Self-Assembled Monolayers of Azobenzene and Alkane Thiols

Surface-bound azobenzenes exhibit reversible photoswitching via trans-cis photoisomerization and have been proposed for a variety of applications such as photowritable optical media, liquid crystal displays, molecular electronics, and smart wetting surfaces. We report a novel synthetic route using simple protection chemistry to form azobenzene-functionalized SAMs on gold and present a mechanistic study of the molecular order, orientation, and conformation in these self-assembled monolayers (SAMs). We use vibrational sum-frequency generation (VSFG) to characterize their vibrational modes, molecular orientation, and photoisomerization kinetics. Trans-cis conformational change of azobenzene leads to the change in the orientation of the nitrile marker group detected by VSFG. Mixed SAMs of azobenzene and alkane thiols are used to investigate the steric hindrance effects. While 100% azobenzene SAMs do not exhibit photoisomerization due to tight packing, we observe reversible switching (>10 cycles) in mixed SAMs with only 34% and 50% of alkane thiol spacers.

Design and evaluation of mixed self-assembled monolayers for a potential use in everolimus eluting coronary stents

Coronary artery disease (CAD) is still the leading cause of death throughout the world. Metal stents are used to widen narrowed arteries. In addition, drug-eluting stents (DES) are widely implanted to decrease the risk of in-stent restenosis. Commercially available polymer-based DES suffer from some limitations. To avoid these drawbacks, the use of self-assembled monolayers (SAMs) in DES has recently been investigated. In this study, methyl- and carboxyl-terminated mixed SAMs on gold (Au) surfaces were successfully prepared. The samples were characterized using scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), Fourier transform infrared spectroscopy (FTIR), and contact angle goniometry (CA). The mixed SAM-coated surfaces were evaluated for everolimus delivery. The drug release in PBS was studied using high performance liquid chromatography (HPLC) and quartz crystal nanobalance (QCN). The results were compared with those related to homogenous SAM-coated surfaces. Significant differences (p<0.05) were found in the amount of drug eluted between the mixed SAM and the homogenous one. The findings are promising for the application of mixed SAMs in DES.

Ultrasensitive impedimetric lectin biosensors with efficient antifouling properties applied in glycoprofiling of human serum samples.

Ultrasensitive impedimetric lectin biosensors recognizing different glycan entities on serum glycoproteins were constructed. Lectins were immobilized on a novel mixed self-assembled monolayer containing 11-mercaptoundecanoic acid for covalent immobilization of lectins and betaine terminated thiol to resist nonspecific interactions. Construction of biosensors based on Concanavalin A (Con A), Sambucus nigra agglutinin type I (SNA), and Ricinus communis agglutinin (RCA) on polycrystalline gold electrodes was optimized and characterized with a battery of tools including electrochemical impedance spectroscopy, various electrochemical techniques, quartz crystal microbalance (QCM), Fourier transform infrared (FT-IR) spectroscopy, atomic force microscopy (AFM), and X-ray photoelectron spectroscopy (XPS) and compared with a protein/lectin microarray. The lectin biosensors were able to detect glycoproteins from 1 fM (Con A), 10 fM (Ricinus communis agglutinin (RCA), or 100 fM (SNA) with a linear range spanning 6 (SNA), 7 (RCA), or 8 (Con A) orders of magnitude. Furthermore, a detection limit for the Con A biosensor down to 1 aM was achieved in a sandwich configuration. A nonspecific binding of proteins for the Con A biosensor was only 6.1% (probed with an oxidized invertase) of the signal toward its analyte invertase and a negligible nonspecific interaction of the Con A biosensor was observed in diluted human sera (1000×), as well. The performance of the lectin biosensors was finally tested by glycoprofiling of human serum samples from healthy individuals and those having rheumatoid arthritis, which resulted in a distinct glycan pattern between these two groups.

Surface-Tethered Iterative Carbohydrate Synthesis: A Spacer Study

Comparative study of Surface-Tethered Iterative Carbohydrate Synthesis (STICS) using HPLC-assisted experimental setup clearly demonstrates benefits of using longer spacer-anchoring systems. The use of mixed self-assembled monolayers helps provide the required space for glycosylation reaction around the immobilized glycosyl acceptor. Both extension of the spacer length and using mixed self-assembled monolayers help promote the reaction, and the beneficial effects may include moving the glycosyl acceptor further out into solution and providing additional conformational flexibility. It is possible that surface-immobilized glycosyl acceptors with a longer spacer (C8-O-C8)-lipoic acid have a higher tendency to mimic a solution-phase reaction environment than acceptors with shorter spacers.

Electroactive mixed self-assembled monolayers: Lateral interactions model updated to interactions between redox and non-redox species

The lateral interactions model, dedicated to random and non-random distributed electroactive species on redox responsive self-assembled monolayers (SAM), was extended to interactions between redox and non-redox species. This approach supports an unusual result achieved in the field of electrochemical transduction without covalent links between redox and complexant units in mixed SAM.

Interactions between Exogenous FGF-2 and Sulfonic Groups: in Situ Characterization and Impact on the Morphology of Human Adipose-Derived Stem Cells

FGF-2 is often used as a supplement to stem cells culture medium aiming at preserving their self-renewal capacity and plasticity through the passages. However, little is known on the influence of the underlying substrate in these interactions. In this study, we have used mixed self-assembled monolayers with different ratios of -SO3H and -OH tail groups to investigate the influence of substrate properties (e.g., charge) on the FGF-2 adsorption and activity. QCM-D data demonstrated that, in the presence of -OH groups, the quantity of the adsorbed FGF-2 is proportional to the percentage of surface -SO3H groups. The bioactivity of the adsorbed FGF-2 follows the same tendency as demonstrated by its interactions with anti-FGF-2. Surprisingly, the adlayer of FGF-2 formed on the surface containing only SO3H-tailed SAMs was similar to the surface with 25% of -SO3H groups, demonstrating that FGF-2 adsorption is not solely driven by electrostatic interactions. We related these results with changes in the morphology of adipose-derived stem cells (ASCs) cultured on the same surfaces.

Irradiation Promoted Exchange Reaction with Disulfide Substituents

Exchange reaction between the primary self-assembled monolayer (SAM) on gold and potential molecular substituents capable of forming a SAM on the same substrate can be promoted by electron irradiation. Here we demonstrate that such a promoted reaction can be performed not only with thiols but with disulfides as substituents as well. This extends significantly the assortments of the suitable compounds, resulting in a broader variety of mixed SAMs and chemical patterns which can be fabricated by this technique. The kinetics of the promoted exchange reaction was studied in detail. The feasibility and practical usefulness of the approach were demonstrated by the experiments with a disulfide substituent bearing a tail group which can serve as an initiator for surface-initiated polymerization. A variety of complex polymer brush patterns was prepared using several representative polymers, relevant for biomedical research and applications, as test systems.

Electrochemical characterization of dehaloperoxidase adsorbates on COOH/OH mixed self-assembled monolayers

Electrochemical characterization of Amphitrite ornata dehaloperoxidase, a monomeric hemoglobin, adsorbed on COOH/OH-terminated alkanethiolate mixed self-assembled monolayers (SAMs) is reported. Adsorption was achieved by simple electrode exposure to low ionic strength protein solution at pH 6.0, and cyclic voltammetry (CV) was employed to measure surface concentration, electron transfer rate, and formal potential under anaerobic conditions. Surface concentration values determined by CV were found to be inversely proportional to scan rate, a behavior that is different than that observed for electron transfer proteins such as cytochrome c. We attribute this behavior to a dynamic heterogeneous adsorbate layer that permits some fraction of the molecules to undergo reorientation during a CV scan. A model was proposed that classifies adsorbates among three populations with respect to their most stable orientations, namely, electroactive adsorbates, latent electroinactive adsorbates capable of undergoing reorientation to electroactive states, and electroinactive adsorbates. Electrochemical results and the proposed model are discussed in terms of the tertiary structure and surface properties of dehaloperoxidase and are compared to the docking and electron transfer reactions of the myoglobin/cytochrome b5 system. Examination of the effects of ionic strength and mixed SAM composition provide support for the proposed model.

Tailored (Bio)Interfaces via Surface Initiated Polymerization: Control of Grafting Density and New Responsive Diblock Copolymer Brushes

SummaryPolymer brush synthesis by surface initiated polymerization (SIP) comprises a powerful strategy to tailor interfacial properties. Among others, the passivation of solid substrates against non‐specific protein adsorption or the implementation of stimuli responsive properties can be achieved. In this context controlled radical polymerization approaches are frequently employed. Here we report on our efforts in the synthesis of oligo and di(ethylene glycol) methylether methacrylate (OEGMA and DEGMA)‐based brushes with (i) controlled grafting density and (ii) new diblock copolymer structures. We assembled mixed self‐assembled monolayers (SAMs) comprising octadecanethiol (ODT), 16‐mercaptohexadecanoic (MHDA) and ω‐mercaptoundecyl bromoisobutyrate (MUBiB) on gold to initiate the controlled radical polymerization of OEGMA and DEGMA. The SAMs as well as the polymer layers obtained by atom transfer radical polymerization for a constant polymerization time were characterized by contact angle measurements with water, FTIR spectroscopy, ellipsometry and atomic force microscopy (AFM). It was found that ODT and MHDA assemble in the mixed SAMs preferentially and despite the different endgroups very similar brushes were obtained. SAMs with fractional surface coverages of the initiator ω‐mercaptoundecyl bromoisobutyrate of &gt; 0.45 showed no further increase in brush thickness, which is in line with a mushroom to brush transition below this coverage. In addition, diblock copolymer brushes comprising POEGMA and poly(tert‐butyl acrylate) (PtBA) blocks were obtained for the first time. The PtBA block was successfully hydrolyzed to yield POEGMA‐block‐poly acrylic acid (PAA) brushes.

Surface-Engineered Growth of AgIn5S8 Crystals

The growth of semiconductor crystals and thin films plays an essential role in industry and academic research. Considering the environmental damage caused by energy consumption during their fabrication, a simpler and cheaper method is desired. In fact, preparing semiconductor materials at lower temperatures using solution chemistry has potential in this research field. We found that solution chemistry, the physical and chemical properties of the substrate surface, and the phase diagram of the multicomponent compound semiconductor have a decisive influence on the crystal structure of the material. In this study, we used self-assembled monolayers (SAMs) to modify the silicon/glass substrate surface and effectively control the density of the functional groups and surface energy of the substrates. We first employed various solutions to grow octadecyltrichlorosilane (OTS), 3-mercaptopropyl-trimethoxysilane (MPS), and mixed OTS-MPS SAMs. The surface energy can be adjusted between 24.9 and 50.8 erg/cm(2). Using metal sulfide precursors in appropriate concentrations, AgIn5S8 crystals can be grown on the modified substrates without any post-thermal treatment. We can easily adjust the nucleation in order to vary the density of AgIn5S8 crystals. Our current process can achieve AgIn5S8 crystals of a maximum of 1 μm in diameter and a minimum crystal density of approximately 0.038/μm(2). One proof-of-concept experiment demonstrated that the material prepared from this low temperature process showed positive photocatalytic activity. This method for growing crystals can be applied to the green fabrication of optoelectronic materials.

Controlled pH Stability and Adjustable Cellular Uptake of Mixed-Charge Nanoparticles

Nanoparticles functionalized with mixed self-assembled monolayers (m-SAMs) comprising positively and negatively charged thiols are stable at both low and high pH but precipitate sharply at the pH where the charges on the particle are balanced (pH(prec)). By adjusting the proportion of the positively and negatively charged ligands in the m-SAM or changing particle size, pH(prec) can be varied flexibly between ~4 and ~7. In addition, changes in the SAMs' composition and particles' net charge translate into different degrees of cellular uptake. Remarkably, the presence of the positively charged thiols allows for the uptake of particles having net negative charge.

Strategies for the Immobilization of Bacteriophages on Gold Surfaces Monitored by Surface Plasmon Resonance and Surface Morphology

The use of bacteriophages as recognition elements for biosensing techniques has recently provoked much interest. Surface plasmon resonance, scanning electron microscopy, and atomic force microscopy were used for the real-time monitoring of the attachment of methicillin-resistant Staphylococcus aureus (MRSA) bacteriophages to gold using several immobilization methods. The MRSA bacterial capture efficiency of phage-functionalized surfaces was studied. We found that whereas the physisorption of phages to gold surfaces affects their biofunctionality, as expressed by their lysing efficiency of bacteria, phages bound via mixed self-assembled monolayers of l-cysteine and 11-mercaptoundecanoic acid permitted both the recognition and disruption of bacterial membranes. This is due to the formation of uniform islands on the gold surfaces, permitting an oriented positioning of the phages, thus better exposing their recognition proteins.

Label-free detection of glycoproteins by the lectin biosensor down to attomolar level using gold nanoparticles

We present here an ultrasensitive electrochemical biosensor based on a lectin biorecognition capable to detect concentrations of glycoproteins down to attomolar (aM) level by investigation of changes in the charge transfer resistance (Rct) using electrochemical impedance spectroscopy (EIS). On polycrystalline gold modified by an aminoalkanethiol linker layer, gold nanoparticles were attached. A Sambucus nigra agglutinin was covalently immobilised on a mixed self-assembled monolayer formed on gold nanoparticles and finally, the biosensor surface was blocked by poly(vinyl alcohol). The lectin biosensor was applied for detection of sialic acid containing glycoproteins fetuin and asialofetuin. Building of a biosensing interface was carefully characterised by a broad range of techniques such as electrochemistry, EIS, atomic force microscopy, scanning electron microscopy and surface plasmon resonance with the best performance of the biosensor achieved by application of HS–(CH2)11–NH2 linker and gold nanoparticles with a diameter of 20nm. The lectin biosensor responded to an addition of fetuin (8.7% of sialic acid) with sensitivity of (338±11) Ωdecade−1 and to asialofetuin (≤0.5% of sialic acid) with sensitivity of (109±10) Ωdecade−1 with a blank experiment with oxidised asialofetuin (without recognisable sialic acid) revealing sensitivity of detection of (79±13) Ωdecade−1. These results suggest the lectin biosensor responded to changes in the glycan amount in a quantitative way with a successful validation by a lectin microarray. Such a biosensor device has a great potential to be employed in early biomedical diagnostics of diseases such as arthritis or cancer, which are connected to aberrant glycosylation of protein biomarkers in biological fluids.

ChemInform Abstract: Catalytic Self‐Assembled Monolayers on Gold Nanoparticles

AbstractReview: 42 refs.

Autocatalytic effect of amine-terminated precursors in mixed self-assembled monolayers

We investigated the formation of mixed self-assembled monolayers (SAMs), comprising two silanes terminated with an amine and a non-reactive functional group, to demonstrate the autocatalysis of the mixed SAMs by the amine-terminated precursors. Measurements of surface energy and angle-resolved X-ray photoelectron spectroscopy on the mixed SAMs revealed that the final composition and the surface coverage of the mixed SAMs after deposition strongly depended on the presence and the concentration of the aminesilane precursor in solution. To explain the observed dependence, we further suggested a mechanism based on the autocatalytic effect of the aminesilane precursor on the mixed SAM. These results highlight the complex role of the aminesilane, which behaves simultaneously as a component of the mixed SAM and a catalyst. An important implication of this is that the compositions of mixed SAMs can be significantly influenced by the autocatalytic effect of the component carrying a reactive functional group such as amine.

Split Aptamer-Based Liquid Crystal Biosensor for ATP Assay

A novel liquid crystal (LC) biosensor based on the recombination of split aptamer chip was developed for the detection of adenosine triphosphate (ATP). One glass slide of the LC cell is first modified with the TEA/DMOAP mixed self-assembled monolayer (SAM) to ensure the homeotropic alignment of LC molecules and the black background of LC cell optical image under the crossed polarized light. The single-strand ATP aptamer is split into two fragments in this method. One of which, as capture probe, is covalently immobilized on the SAM, while another is used as the detection probe. In the presence of ATP, those two fragments will be combined with each other and exhibit the same role as the ATP aptamer. This binding event leads to a great enhancement in the optical signal of the LC biosensor due to the space size from small to big, which can effectively disrupt the orientational arrangement of LCs, resulting in the corresponding changes of optical images under the crossed polarized light. But in the absence of ATP, those two fragments can not be combined with each other, and as a result, there is no orientational response of LCs and the optical image under the crossed polarized light is still black. So the LC-based imaging method has a sensitive and clear distinction between positive and negative results. On the basis of such an inhibition mechanism the LC biosensor can be used as an effective way to realize the detection of ATP. When the concen- tration of analyte ATP below a critical value (10 nmol/L), there is no clear change in the optical image. The results showed that the detection limit of ATP is 10 nmol/L. This study provides a simple and sensitive ATP LC biosensing approach and offers effective strategies for the development of small molecules LC biosensors. Keywords liquid crystal; self-assembled film; biosensor; ATP; aptamer

Photoresponse enhancement by mixing of an alcohol-soluble C60 derivative into a ruthenium complex monolayer

We have investigated the effect of mixing an alcohol-soluble C60 derivative into a self-assembled monolayer (SAM), which consisted of a redox-active Ru-complex with multipoint anchoring groups, on an indium tin oxide surface. Angle-resolved X-ray photoelectron spectroscopy of the mixed SAM revealed that the C60 derivative was well incorporated into the redox-active Ru-complex SAM. In addition, some of the C60 derivatives were present on the mixed SAM surface. In the presence of a sacrificial reagent, the action spectra of the mixed molecular layer exhibited a broad spectral response due to the presence of the C60 derivative, whereas a sharp response was observed for the monocomponent Ru-complex SAM. We propose that an efficient charge separation arising from the combination of the C60 derivative and the Ru-complex enhanced the spectral response of the mixed SAM.

Hetero-Coated Magnetic Microcarriers for Point-Of-Care Diagnostics

SU8 microcarriers containing magnetic thin film (40 nm cobalt) elements were fabricated by photolithography and thermal evaporation, and included a gold coating on the base. The microcarriers, which can be encoded with a magnetic barcode for later identification in a multiplexed suspension assay, now possess two distinct surfaces for further chemical derivitization. This allows us to attach different bio-molecules to each side, which we suggest, could be used to add a positive control to typical hybridization assays as a test for binding conditions, thus giving confidence against false negative events. As a proof-of-concept we developed a chemical pathway to attach <formula formulatype="inline" xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"><tex Notation="TeX">$5^{\prime},6^{\prime}$</tex></formula> -tetramethyl rhodamine onto the gold coated side via the growth of a mixed self-assembled monolayer (characterized by Electrochemical Quartz Crystal Microbalance measurements), while also attaching fluorescein to the SU8 side after first adding a homo-bifunctional spacer to the native surface epoxide groups. Fluorescence microscopy was used to verify the success of this strategy and the feasibility of using PicoGreen as a post-hybridization reporter was studied.

Using A Fiber Optic Particle Plasmon Resonance Biosensor To Determine Kinetic Constants of Antigen–Antibody Binding Reaction

In this paper, one simple and label-free biosensing method has been developed for determining the binding kinetic constants of antiovalbumin antibody (anti-OVA) and anti-mouse IgG antibody using the fiber optic particle plasmon resonance (FOPPR) biosensor. The FOPPR sensor is based on gold-nanoparticle-modified optical fiber, where the gold nanoparticle surface has been modified by a mixed self-assembled monolayer for conjugation of a molecular probe reporter (ovalbumin or mouse IgG) to dock with the corresponding analyte species such as anti-OVA or anti-mouse IgG. The binding process, occurring when an analyte reacts with a probe molecule immobilized on the optical fiber, can be monitored in real-time. In addition, by assuming a Langmuir-type adsorption isotherm to measure the initial binding rate, the quantitative determination of binding kinetic constants, the association and dissociation rate constants, yields k(a) of (7.21 ± 0.4) × 10(3) M(-1) s(-1) and k(d) of (2.97 ± 0.1) × 10(-3) s(-1) for OVA/anti-OVA and k(a) of (1.45 ± 0.2) × 10(6) M(-1) s(-1) and k(d) of (2.97 ± 0.6) × 10(-2) s(-1) for mouse IgG/anti-mouse IgG. We demonstrate that the FOPPR biosensor can study real-time biomolecular interactions.