Self-assembled monolayers (SAMs) of alkanethiolates on gold have become an important tool for probing cell-material interactions. Emerging studies in stem cell biology are particularly reliant on well-defined model substrates, and rapid and highly controllable fabrication methods may be necessary to characterize the wide array of stem cell-material interactions. Therefore, this study describes a rapid method to create SAM cell culture substrates with multiple discrete regions of controlled peptide identity and density. The approach uses an NaBH4 solution to selectively remove regions of bio-inert, hydroxyl-terminated oligo(ethylene glycol) alkanethiolate SAM, then locally replace them with mixed SAMs of hydroxyl- and carboxylic acid-terminated oligo(ethylene glycol) alkanethiolates. The cell adhesion peptide Arg-Gly-Asp-Ser-Pro (RGDSP) was then covalently linked to carboxylic acid-terminated mixed SAM regions to create cell adhesive environments within a bio-inert background. SAM preparation and peptide immobilization were characterized using polarization modulation–infrared reflection-absorption spectroscopy (PMIRRAS), as well as assays to monitor conjugation of a fluorescently-labeled peptide. This “localized SAM replacement” method was achieved using an array of microchannels, which facilitated rapid and simple processing. Results indicate that immobilized RGDSP promoted spatially localized attachment of human mesenchymal stem cells (hMSCs) within specified regions, while maintaining a stable, bio-inert background in serum-containing cell culture conditions for up to 14 days. Cell attachment to patterned regions presenting a range of cell adhesion peptide densities demonstrated that peptide identity and density strongly influence hMSC spreading and focal adhesion density. These substrates contain discrete, well-defined microenvironments for stem cell culture, which could ultimately enable high-throughput screening for the effects of immobilized signals on stem cell phenotype.
Two series of self-assembled monolayers (SAMs) of ω-substituted alkanethiolates on gold were used to systematically examine the effects of varying substratum surface chemistry and energy on the attachment of two model organisms of interest to the study of marine biofouling, the bacterium Cobetia marina (formerly Halomonas marina) and zoospores of the alga Ulva linza (formerly Enteromorpha linza). SAMs were formed on gold-coated glass slides from solutions containing mixtures of methyl- and carboxylic acid-terminated alkanethiols and mixtures of methyl- and hydroxyl-terminated alkanethiols. C. marina attached in increasing numbers to SAMs with decreasing advancing water contact angles (θAW), in accordance with equation-of-state models of colloidal attachment. Previous studies of Ulva zoospore attachment to a series of mixed methyl- and hydroxyl-terminated SAMs showed a similar correlation between substratum θAW and zoospore attachment. When the hydrophilic component of the SAMs was changed to carboxylate, however, the profile of attachment of Ulva was significantly different, suggesting that a more complex model of interfacial energetics is required.
In this research, nanoimprint lithography (NIL) was used for patterning crystalline zinc oxide (ZnO) nanorods on the silicon substrate. To fabricate nano-patterned ZnO nanorods, patterning of an n-octadecyltrichlorosilane (OTS) self-assembled monolayers (SAMs) on SiO2 substrate was prepared by the polymer mask using NI. The ZnO seed layer was selectively coated only on the hydrophilic SiO2 surface, not on the hydrophobic OTS SAMs surface. The substrate patterned with the ZnO seed layer was treated with the oxygen plasma to oxidize the silicon surface. It was found that the nucleation and initial growth of the crystalline ZnO were proceeded only on the ZnO seed layer, not on the silicon oxide surface. ZnO photoluminescence spectra showed that ZnO nanorods grown from the seed layer treated with plasma showed lower intensity than those untreated with plasma at 378 nm, but higher intensity at 605 nm. It is indicated that the seed layer treated with plasma produced ZnO nanorods that had a more oxygen vacancy than those grown from seed layer untreated with plasma. Since the oxygen vacancies on ZnO nanorods serve as strong binding sites for absorption of various organic and inorganic molecules. Consequently, a nano-patterning of the crystalline ZnO nanorods grown from the seed layer treated with plasma may give the versatile applications for the electronics devices.
The cell-material interaction is a complex bi-directional and dynamic process that mimics to a certain extent the natural interactions of cells with the extracellular matrix. Cells tend to adhere and rearrange adsorbed extracellular matrix (ECM) proteins on the material surface in a fibril-like pattern. Afterwards, the ECM undergoes proteolytic degradation, which is a mechanism for the removal of the excess ECM usually approximated with remodeling. ECM remodeling is a dynamic process that consists of two opposite events: assembly and degradation.
This work investigates matrix protein dynamics on mixed self-assembled monolayers (SAMs) of –OH and –CH3 terminated alkanethiols. SAMs assembled on gold are highly ordered organic surfaces able to provide different chemical functionalities and well-controlled surface properties. Fibronectin (FN) was adsorbed on the different surfaces and quantified in terms of the adsorbed surface density, distribution and conformation. Initial cell adhesion and signaling on FN-coated SAMs were characterized via the formation of focal adhesions, integrin expression and phosphorylation of FAKs. Afterwards, the reorganization and secretion of FN was assessed. Finally, matrix degradation was followed via the expression of matrix metalloproteinases MMP2 and MMP9 and correlated with Runx2 levels. We show that matrix degradation at the cell material interface depends on surface chemistry in MMP-dependent way.
This work provides a broad overview of matrix remodeling at the cell-material interface, establishing correlations between surface chemistry, FN adsorption, cell adhesion and signaling, matrix reorganization and degradation. The reported findings improve our understanding of the role of surface chemistry as a key parameter in the design of new biomaterials. It demonstrates the ability of surface chemistry to direct proteolytic routes at the cell-material interface, which gains a distinct bioengineering interest as a new tool to trigger matrix degradation in different biomedical applications.
In this report, alkanethiol self assembled monolayers (SAM) with two different chain lengths were used to immobilize the functionalizing enzyme (glucose oxidase) onto gold nanopillar modified electrodes and the electrochemical processes of these functionalized electrodes in glucose detection were investigated. First, the formation of these SAMs on the nanopillar modified electrodes was characterized by the cyclic voltammetry and electrochemical impedance spectroscopy techniques, and then the detection sensitivity of these functionalized electrodes to glucose was evaluated by the amperometry technique. Results showed that the SAM of alkanethiols with a longer chain length resulted in a higher degree of surface coverage with less defect and a higher electron transfer resistance, whereas the SAM of alkanethiols with a shorter chain length gave rise to a higher detection sensitivity to glucose. This study sheds some new insight into how to enhance the sensing performance of nanopillar modified electrodes.
Gold nanopillar modified electrodes; self assembled monolayer; alkanethiols; electrochemical processes; glucose detection; biosensors
Substrate-mediated gene delivery describes the immobilization of gene therapy vectors to a biomaterial, which enhances gene transfer by exposing adhered cells to elevated DNA concentrations within the local microenvironment. Surface chemistry has been shown to affect transfection by nonspecifically immobilized complexes using self-assembled monolayers (SAMs) of alkanethiols on gold. In this report, SAMs were again used to provide a controlled surface to investigate whether the presence of oligo(ethylene glycol) (EG) groups in a SAM could affect complex morphology and enhance transfection. EG groups were included at percentages that did not affect cell adhesion. Nonspecific complex immobilization to SAMs containing combinations of EG- and carboxylic acid-terminated alkanethiols resulted in substantially greater transfection than surfaces containing no EG groups or SAMs composed of EG groups combined with other functional groups. Enhancement in transfection levels could not be attributed to complex binding densities or release profiles. Atomic force microscopy imaging of immobilized complexes revealed that EG groups within SAMs affected complex size and appearance and could indicate the ability of these surfaces to preserve complex morphology upon binding. The ability to control the morphology of the immobilized complexes and influence transfection levels through surface chemistry could be translated to scaffolds for gene delivery in tissue engineering and diagnostic applications.
Gene delivery; Reverse transfection; Atomic force microscopy (AFM); Self-assembled monolayers
Gene transfer has many potential applications in basic and applied sciences. In vitro, DNA delivery can be enhanced by increasing the concentration of DNA in the cellular microenvironment through immobilization of DNA to a substrate that supports cell adhesion. Substrate-mediated delivery describes the immobilization of DNA, complexed with cationic lipids or polymers, to a biomaterial or substrate. As surface properties are critical to the efficiency of the surface delivery approach, self-assembled monolayers (SAMs) of alkanethiols on gold were used to correlate surface chemistry of the substrate to binding, release, and transfection of non-specifically immobilized complexes. Surface hydrophobicity and ionization were found to mediate both DNA complex immobilization and transfection, but had no effect on complex release. Additionally, SAMs were used in conjunction with soft lithographic techniques to imprint substrates with specific patterns, resulting in patterned DNA complex deposition and transfection, with transfection efficiencies in the patterns nearing 40%. Controlling the interactions between complexes and substrates, with the potential for patterned delivery, can be used to locally enhance or regulate gene transfer, with applications to tissue engineering scaffolds and transfected cell arrays.
Self-assembled monolayers; Gene delivery; Reverse transfection; Solid-phase delivery; Substrate mediated
This paper reports the development of a class of isoform-selective peptide substrates for measuring endogenous lysine deacetylase (KDAC) activities in cell culture. The peptides were first identified by comparing the substrate specificity profiles of the four KDAC isoforms KDAC2, KDAC3, KDAC8, and sirtuin 1 (SIRT1) on a 361-member hexapeptide array wherein the two C-terminal residues to the acetylated lysine were varied. The arrays were prepared by immobilizing the peptides to a self-assembled monolayer of alkanethiolates on gold and could therefore be analyzed by a mass spectrometry technique termed SAMDI (self-assembled monolayers for matrix assisted laser desorption/ionization time-of-flight mass spectrometry). Arrays presenting the selective substrates were treated with nuclear extracts from HeLa, Jurkat, and smooth muscle cells and analyzed to measure endogenous deacetylase activities. We then use the arrays to profile KDAC activity through the HeLa cell cycle. We find that the activity profile of the KDAC3 selective peptide closely mirrors the changing acetylation state of the H4 histone, suggesting a role for this enzyme in cell cycle regulation. This work is significant because it describes a general route for identifying selective substrates that can be used to understand the differential roles of members of the deacetylase enzyme family in complex biological processes and further because the label-free approach avoids perturbation of enzyme activity that has plagued fluorescence-based assays.
Fibrinogen (Fbg) mediates platelet aggregation through its binding to the αIIbβ3 integrin receptor. Despite the many studies of platelet aggregation and blood clotting, the interaction of Fbg with the platelet integrin has remained unresolved. This paper reports on the use of self-assembled monolayers (SAMs) of alkanethiolates on gold to study the adhesion of αIIbβ3 CHO K1 cells to the GRGDS and HHLGGAKQAGDV motifs within Fbg. The peptides were immobilized to a monolayer that otherwise prevented nonspecific interactions of the cells with the substrate. Monolayers presenting GRGDSC or HHLGGAKQAGDVC were effective at mediating αIIbβ3 CHO K1 cell adhesion and spreading and were comparable to the use of Fbg-coated substrates, suggesting that both sequences can bind the receptor independently. A comparison of cell adhesion to several peptide truncations revealed that AGD was the minimal binding sequence in HHLGGAKQAGDV, and inhibition experiments showed that AGD and RGD were competitive ligands for the receptor. A peptide array of GXGDSC peptides revealed that αIIbβ3 CHO K1 cells adhered to peptides containing basic or hydrophobic residues in the X position, revealing the relaxed specificity with which αIIbβ3 recognizes its ligands. This work therefore suggests that AGD and RGD interact with Fbg in a functionally similar manner and that the use of AGD peptides may lead to a new generation of anti-thrombotic agents.
We have used the orthogonal carbodiimide condensation and Copper-catalyzed azide-alkyne “click” cycloaddition (CuAAC) reactions to prepare self-assembled monolayers that present distinct peptides to stem cells in a bio-inert background. The approach involved first forming mixed SAMs with three components: i) an azide-terminated hexaethylene glycol alkanethiolate (HS---EG6---N3), ii) a carboxylate-terminated hexaethylene glycol alkanethiolate (HS---EG6---COOH), and iii) a triethylene glycol alkanethiolate (HS---EG3). An acetylene-bearing peptide and an amine-terminated peptide were then immobilized to these substrates using a “click” CuAAC reaction and a carbodiimide condensation reaction, respectively. Polarization-modulated infrared reflectance-absorbance spectroscopic analysis demonstrated formation of well-ordered, close-packed SAMs, chemoselective conjugation of amine-terminated peptides to surface carboxylate groups, and subsequent conjugation of acetylene-terminated peptides to the azide groups on SAMs. Varying the mole fraction of HS---EG6---N3, HS---EG6---COOH, and HS---EG3 during SAM formation allowed for control over the densities of each peptide on the substrate. Substrates presenting varying surface densities of RGESP (a non-functional peptide), RGDSP (a cell adhesion peptide) or TYRSRKY (a heparin/heparan sulfate-binding peptide) were then used to characterize the relationship between peptide surface density and human mesenchymal stem cell (hMSC) adhesion. Results demonstrate that RGESP does not influence RGDSP-mediated adhesion of hMSCs, which indicates that a second peptide with distinct bio-activity can be immobilized alongside RGDSP to characterize the influence of two peptides on hMSC behavior. Our results also demonstrate that RGDSP and TYRSRKY act synergistically to promote hMSC adhesion in the absence of serum. Interestingly, heparin sequestered by TYRSRKY inhibits cell adhesion on substrates presenting RGDSP = 0.1% and > 0.1% TYRSRKY or RGDSP = 1% and > 0.5% TYRSRKY. Taken together, these results indicate that two peptides can be controllably presented to stem cells on the same otherwise bio-inert SAM substrate, and that multiple, distinct extracellular moieties act in concert to regulate hMSC adhesion.
High-density live cell array serves as a valuable tool for the development of high-throughput immunophenotyping systems and cell-based biosensors. In this paper, we have, for the first time, demonstrated a simple fabrication process to form the hexamethyldisilazane (HMDS) and poly(ethylene glycol) (PEG) binary molecular surface which can be used to effectively form high fidelity cell arrays. The HMDS self-assembled monolayer (SAM) on glass substrates was photolithographically patterned and its ability to physically adsorb proteins was characterized by contact angle measurement and fluorescence microscopy respectively. Passivation of the non-HMDS coated background by PEG was verified to have no impact on the pre-patterned HMDS and greatly inhibited the non-specific protein binding. Using the biotin–streptavidin complexation as an intermediate, uniform orientation and high bioactivity were achieved for the immobilized B lymphocyte specific anti-CD19 antibodies and therefore ensured the formation of high resolution B lymphocyte arrays. The cell–ligand interaction specificity was investigated and the anti-CD19 decorated micropatterns presented a much higher cell-capturing rate (88%) than those modified by non-specific ligands (15% for anti-CD5 and 7% for streptavidin). The approach was verified to be biocompatible and the properties of the antibody-modified surface were maintained after 12 h cell culture. The HMDS monolayer formation and patterning processes, and the universal HMDS/biotin-BSA/streptavidin template, provide a very simple and convenient process to generate high resolution micropatterns of cell-adhesive ligands and are extendable to form arrays of other types of cells as well.
Controllers for scanning probe instruments can be programmed for automated lithography to generate desired surface arrangements of nanopatterns of organic thin films, such as n-alkanethiol self-assembled monolayers (SAMs). In this report, atomic force microscopy (AFM) methods of lithography known as nanoshaving and nanografting are used to write nanopatterns within organic thin films. Commercial instruments provide software to control the length, direction, speed, and applied force of the scanning motion of the tip. For nanoshaving, higher forces are applied to an AFM tip to selectively remove regions of the matrix monolayer, exposing bare areas of the gold substrate. Nanografting is accomplished by force-induced displacement of molecules of a matrix SAM, followed immediately by the surface self-assembly of n-alkanethiol molecules from solution. Advancements in AFM automation enable rapid protocols for nanolithography, which can be accomplished within the tight time restraints of undergraduate laboratories. Example experiments with scanning probe lithography (SPL) will be described in this report that were accomplished by undergraduate students during laboratory course activities and research internships in the chemistry department of Louisiana State University. Students were introduced to principles of surface analysis and gained “hands-on” experience with nanoscale chemistry.
Bacterial biofilms cause serious problems, such as antibiotic resistance and medical device-related infections. To further understand bacterium-surface interactions and to develop efficient control strategies, self-assembled monolayers (SAMs) of alkanethiols presenting different functional groups on gold films were analyzed to determine their resistance to biofilm formation. Escherichia coli was labeled with green florescence protein, and its biofilm formation on SAM-modified surfaces was monitored by confocal laser scanning microscopy. The three-dimensional structures of biofilms were analyzed with the COMSTAT software to obtain information about biofilm thickness and surface coverage. SAMs presenting methyl, l-gulonamide (a sugar alcohol tethered with an amide bond), and tri(ethylene glycol) (TEG) groups were tested. Among these, the TEG-terminated SAM was the most resistant to E. coli biofilm formation; e.g., it repressed biofilm formation by E. coli DH5α by 99.5% ± 0.1% for 1 day compared to the biofilm formation on a bare gold surface. When surfaces were patterned with regions consisting of methyl-terminated SAMs surrounded by TEG-terminated SAMs, E. coli formed biofilms only on methyl-terminated patterns. Addition of TEG as a free molecule to growth medium at concentrations of 0.1 and 1.0% also inhibited biofilm formation, while TEG at concentrations up to 1.5% did not have any noticeable effects on cell growth. The results of this study suggest that the reduction in biofilm formation on surfaces modified with TEG-terminated SAMs is a result of multiple factors, including the solvent structure at the interface, the chemorepellent nature of TEG, and the inhibitory effect of TEG on cell motility.
This paper describes a model system for studying the auto-catalytic phosphorylation of an immobilized substrate by a kinase enzyme. This work uses self-assembled monolayers (SAMs) of alkanethiolates on gold to present the peptide substrate on a planar surface. Treatment of the monolayer with Abl kinase results in phosphorylation of the substrate. The phosphorylated peptide then serves as a ligand for the SH2 adaptor domain of the kinase and thereby directs the kinase activity to nearby peptide substrates. This directed reaction is intramolecular and proceeds with a faster rate than does the initial, intermolecular reaction, making this an auto-catalytic process. The kinetic non-linearity gives rise to properties that have no counterpart in the corresponding homogeneous phase reaction: in one example, the rate for phosphorylation of a mixture of two peptides is faster than the sum of the rates for phosphorylation of each peptide when presented alone. This work highlights the use of an adaptor domain in modulating the activity of a kinase enzyme for an immobilized substrate and offers a new approach for studying biochemical reactions in spatially inhomogeneous settings.
Adaptor domain; autocatalysis; interfacial; SAMDI; phosphorylation
Self-assembled monolayers (SAMs) of nitrile-substituted oligo(phenylene ethynylene) thiols (NC-OPEn) with a variable chain length n (n ranging from one to three structural units) on Au(111) were studied by synchrotron-based high-resolution X-ray photoelectron spectroscopy and near-edge absorption fine-structure spectroscopy. The experimental data suggest that the NC-OPEn molecules form well-defined SAMs on Au(111), with all the molecules bound to the substrate through the gold–thiolate anchor and the nitrile tail groups located at the SAM–ambient interface. The packing density in these SAMs was found to be close to that of alkanethiolate monolayers on Au(111), independent of the chain length. Similar behavior was found for the molecular inclination, with an average tilt angle of ~33–36° for all the target systems. In contrast, the average twist of the OPEn backbone (planar conformation) was found to depend on the molecular length, being close to 45° for the films comprising the short OPE chains and ~53.5° for the long chains. Analysis of the data suggests that the attachment of the nitrile moiety, which served as a spectroscopic marker group, to the OPEn backbone did not significantly affect the molecular orientation in the SAMs.
nitrile substitution; oligo(phenylene ethynylene); self-assembled monolayers; twist angle; X-ray absorption spectroscopy
Self-assembled monolayers (SAMs) are widely used to confine proteins and cells to a pattern in order to study cellular processes and behavior. In order to fully explore some of these phenomena, it is necessary to control cell growth and confinement for several weeks. Here we present a simple method by which protein and cellular confinement to a pattern can be maintained for more than 35 days. This represents a significant increase in pattern stability compared to previous monolayer systems and is achieved by using an amide-linked glycol monomer on 50 Å titanium/100 Å gold-coated glass coverslips. In addition, this study provides insight into the method of SAM degradation and excludes interfacial mixing of the monomers and blooming of the adlayer as major mechanisms for SAM degradation.
Self-Assembled Monolayer (SAM); patterned cell culture; monolayer stability; pattern fidelity; microcontact printing
Self-assembled monolayers (SAMs) of alkanethiols (ATs) on gold can be used to fabricate surfaces for nanoscience and biology. The chemical structure of the interface can be tailored simply by modifying the AT head group. To streamline access to different precursor ATs, we developed a general solid-phase synthetic route. A key feature of this route is the use of a modified resin containing an AT-linker (“AT-resin”) because it minimizes purification steps. The precursor to AT-resin was prepared in 5 steps, and all of the synthetic intermediates are stable solids that can be purified by crystallization. Accordingly, the AT-resin can be prepared on a multi-gram scale. The utility of AT-resin was evaluated by using it to generate a variety of ATs. For example, ATs presenting different types of integrin-binding ligands (linear and cyclic RGD derivatives) were prepared and used to form arrays of SAMs that support cell adhesion. Additionally, AT-resin also provides a starting point for the synthesis of ATs presenting reactive groups (e.g., an amine-reactive AT or a maleimide-containing alkanedisulfide) or protein immobilization tags (e.g., biotin-AT). Thus, our synthetic strategy provides a convenient and flexible means for the synthesis of the necessary building blocks for custom SAMs and SAM arrays.
This talk describes an approach to using mass spectrometry to analyze biochips—including enzyme-mediated reaction of immobilized biomolecules and protein-protein interactions. The method is based on self-assembled monolayers of alkanethiolates on gold that present proteins and small molecules with control over the densities, patterns, and orientations of these species. Of particular interest is the development of fusion protein capture strategies that give selective and covalent immobilization of proteins. In one example, the serine esterase cutinase reacts with phosphonate capture ligands to give an active-site covalent adduct. Application of a cutinase fusion protein with a monolayer presenting the capture ligand results in immobilization of the display protein, with strict control over both the orientation and the density of this reagent. The chips are compatible with matrix-assisted laser desorption ionization mass spectrometry, and therefore do not require fluorescent or radioisotopic labels for analysis. This technique, termed SAMDI MS, can efficiently monitor a broad class of enzyme activities, including kinase, protease, methyltransferase, and carbohydrate-directed modifications, and can detect proteins having molecular weights up to 100 kDa. The talk will describe examples in assays of endogeneous cellular activities and protein-protein interaction mapping.
Bioactive glass (BG) can directly bond to living bone without fibrous tissue encapsulation. Key mechanistic steps of BG’s activity are attributed to calcium phosphate formation, surface hydroxylation and fibronectin (FN) adsorption. In the present study, self-assembled monolayers (SAMs) of alkanesilanes with different surface chemistry (OH, NH2, and COOH) were used as a model system to mimic BG’s surface activity. Calcium phosphate (Ca-P) was formed on SAMs by immersion in a solution which simulates the electrolyte content of physiological fluids. FN adsorption kinetics and monolayer coverage was determined on SAMs with or without Ca-P coating. The surface roughness was also examined on these substrates before and after FN adsorption. The effects of FN-adsorbed, Ca-P coated SAMs on the function of MC3T3-E1 were evaluated by cell growth, expression of alkaline phosphatase activity, and actin cytoskeleton formation. We demonstrate that, although the FN monolayer coverage and the rms roughness are similar on −OH and −COOH terminated SAMs with or without Ca-P coating, higher levels of ALP activity, more actin cytoskeleton formation and more cell growth are obtained on −OH and −COOH terminated SAMs with Ca-P coating. In addition, although the FN monolayer coverage is higher on Ca-P coated −NH2 terminated SAMs and SiOx surfaces, higher levels of ALP activity and more cell growth are obtained on Ca-P coated −OH and −COOH terminated SAMs. Thus with same Ca-P coatings, different surface functional groups have different effects on the function of osteoblastic cells. These findings represent new insights into the mechanism of bioactivity of BG and, thereby, may lead to designing superior constructs for bone grafting.
self assembled monolayers; calcium phosphate; protein adsorption; cell attachment; proliferation; alkaline phosphatase activity
We report progress towards the surface-enhanced Raman scattering (SERS) characterization of self-assembled monolayers (SAMs) on uniform Ag nanocubes. This study quantifies changes in the SAMs induced by the presence of aqueous glucose. The SAMs were prepared from dodecanethiol and they were representative of highly ordered monolayers as indicated by SERS analysis. We examined the SAMs response to glucose and observed conformational changes in the alkanethiolate SAMs. Analysis of the trans and gauche bands as well as the C-H stretching modes of the SAMs suggest that the analyte-SAM interactions were superficial and there was no penetration for the glucose molecules into the monolayers.
Self-assembled monolayers (SAMs) of pyridylthio-functionalized multiwalled carbon nanotubes (pythio-MWNTs) have been constructed on the gold substrate surface, which were used as a support to immobilize cytochrome c (Cyt c). The assembly processes of the SAMs and adsorption of Cyt c were monitored by using quartz crystal microbalance (QCM). Based on the frequency change of the QCM resonator, the surface coverage for the SAMs of pythio-MWNTs was estimated to be about 5.2 μg/cm2, and that of the Cyt c adsorbed was about 0.29 μg/cm2. For the gold electrode modified by the SAMs of pythio-MWNTs-Cyt c, a quasi-reversible redox wave was recorded with the cathodic and anodic potentials at about −0.55 and −0.28 V vs Ag/AgCl, respectively. Compositions and morphologies of the SAMs before and after immobilization of Cyt c were characterized by X-ray photoelectron spectroscopy, Raman spectroscopy, scanning electron microscopy, and atomic force microscopy.
Carbon nanotube; Self-assembled monolayer; Protein; Adsorption; Morphology
Self-assembled monolayers (SAMs) of alkanethiolates on gold are chemically defined substrates that can be used to evaluate the effects of an immobilized biomolecule. However, the types of biomolecules that can influence stem cell behavior are numerous and inter-related, and efficient experimental formats are a critical need. Here we employed a SAM array technology to investigate the effects of multiple, distinct peptides and peptide combinations on human mesenchymal stem cell (hMSC) behavior. Specifically, we characterized the conjugation of peptide mixtures to SAM arrays and then investigated the combined effects of a bone morphogenic protein receptor-binding peptide (BR-BP), a heparin proteoglycan-binding peptide (HPG-BP), and varied densities of the integrin-binding ligand Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP) on hMSC surface coverage and alkaline phosphatase activity. Results indicate that an amine reactive fluorescent probe can be used to characterize peptide composition after immobilization in SAM array spots. Furthermore, hMSC response to BR-BP and HPG-BP is dependent on GRGDSP density and at day 7, hMSC alkaline phosphatase expression is highly dependent on GRGDSP density. Taken together, we demonstrate how a SAM array approach can be used to probe the combinatorial effects of multiple peptides and motivate further investigations into potential synergies between cell adhesion and other bioactive peptides.
Vascular stents are small tubular scaffolds used in the treatment of arterial stenosis (narrowing of the vessel). Most vascular stents are metallic and are deployed either by balloon expansion or by self-expansion. A shape memory polymer (SMP) stent may enhance flexibility, compliance, and drug elution compared to its current metallic counterparts. The purpose of this study was to describe the fabrication of a laser-activated SMP stent and demonstrate photothermal expansion of the stent in an in vitro artery model.
A novel SMP stent was fabricated from thermoplastic polyurethane. A solid SMP tube formed by dip coating a stainless steel pin was laser-etched to create the mesh pattern of the finished stent. The stent was crimped over a fiber-optic cylindrical light diffuser coupled to an infrared diode laser. Photothermal actuation of the stent was performed in a water-filled mock artery.
At a physiological flow rate, the stent did not fully expand at the maximum laser power (8.6 W) due to convective cooling. However, under zero flow, simulating the technique of endovascular flow occlusion, complete laser actuation was achieved in the mock artery at a laser power of ~8 W.
We have shown the design and fabrication of an SMP stent and a means of light delivery for photothermal actuation. Though further studies are required to optimize the device and assess thermal tissue damage, photothermal actuation of the SMP stent was demonstrated.
A rapid and cost-effective lithographic method, polymer blend lithography (PBL), is reported to produce patterned self-assembled monolayers (SAM) on solid substrates featuring two or three different chemical functionalities. For the pattern generation we use the phase separation of two immiscible polymers in a blend solution during a spin-coating process. By controlling the spin-coating parameters and conditions, including the ambient atmosphere (humidity), the molar mass of the polystyrene (PS) and poly(methyl methacrylate) (PMMA), and the mass ratio between the two polymers in the blend solution, the formation of a purely lateral morphology (PS islands standing on the substrate while isolated in the PMMA matrix) can be reproducibly induced. Either of the formed phases (PS or PMMA) can be selectively dissolved afterwards, and the remaining phase can be used as a lift-off mask for the formation of a nanopatterned functional silane monolayer. This “monolayer copy” of the polymer phase morphology has a topographic contrast of about 1.3 nm. A demonstration of tuning of the PS island diameter is given by changing the molar mass of PS. Moreover, polymer blend lithography can provide the possibility of fabricating a surface with three different chemical components: This is demonstrated by inducing breath figures (evaporated condensed entity) at higher humidity during the spin-coating process. Here we demonstrate the formation of a lateral pattern consisting of regions covered with 1H,1H,2H,2H-perfluorodecyltrichlorosilane (FDTS) and (3-aminopropyl)triethoxysilane (APTES), and at the same time featuring regions of bare SiOx. The patterning process could be applied even on meter-sized substrates with various functional SAM molecules, making this process suitable for the rapid preparation of quasi two-dimensional nanopatterned functional substrates, e.g., for the template-controlled growth of ZnO nanostructures .
breath figure; nanopatterned template; polymer blend lithography (PBL); self-assembled monolayer (SAM); self assembly; spin coating; vapor phase
We have used a Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) “click” reaction to prepare self-assembled monolayers (SAMs) presenting the cell adhesion peptide Arg-Gly-Asp-Ser-Pro (RGDSP) in a bio-inert background. The surface preparation approach involved first forming mixed SAMs with an azide-terminated hexaethylene glycol alkanethiolate (HS---EG6---N3) and a triethylene glycol alkanethiolate (HS---EG3), then using the CuAAC reaction to immobilize an alkyne-terminated peptide. The mixed SAMs were classified as bio-inert, as SAMs comprised of 10 mole percent HS---EG6---N3 and 90 mole percent HS---EG3 showed minimal non-specific protein adsorption in solutions of 1 mg/ml lysozyme or 10% fetal bovine serum. The reaction between an acetylene-terminated peptide and an azide-terminated SAM proceeded rapidly and quantitatively in the presence of a Cu(I)-TBTA complex, displaying pseudo-first order kinetics with a rate constant of ∼ 0.2 min−1. Varying the ratio of HS---EG6---N3 to HS---EG3 during SAM formation allowed for control over the density of azide and, in turn, the density of RGDSP on the substrates. These substrates were therefore used to study the detailed relationship between RGDSP surface density and human mesenchymal stem cell (hMSC) adhesion, spreading, and focal adhesion complex formation, without interference from non-specifically adsorbed serum proteins. Results indicate that an RGDSP intermolecular spacing of 36 nm or less (≥ 0.01 mole percent on the surface) is sufficient for hMSC adhesion and a spacing of 11 nm or less (≥ 0.05 mole percent on the surface) is sufficient for cell spreading and focal adhesion complex formation. In total, our results demonstrate that CuAAC is a suitable mechanism for conjugating peptides to otherwise bio-inert SAMs, and that the resulting SAMs can be used to study the dependence of peptide density on stem cell behavior.