Electrochemical desorption of self-assembled monolayers noninvasively releases patterned cells from geometrical confinements

This report describes a method to pattern mammalian cells using self-assembled monolayers (SAMs), and then to use electrochemical desorption of these monolayers to release cells from their patterns. This method uses an oligo(ethyleneglycol)-terminated SAM to prevent,-and a methyl-terminated SAM to allow-adsorption of proteins and attachment of bovine capillary endothelial cells. Electrochemical removal of the oligo(ethyleneglycol)-terminated SAM allowed proteins to adsorb onto areas that had been previously inert and enabled cells to migrate into these areas. This straightforward technique is useful in bioassays for drug screening and for fundamental studies in cell biology.     

Electrostatic assembly of gold colloidal nanoparticles on organosilane monolayers patterned by microcontact electrochemical conversion

A new approach is introduced for electrostatically guided adsorption of colloidal nanoparticles onto a patterned self-assembled monolayer (SAM) with feature sizes ranging from nm to mm. Patterning of the adsorption templates is realized by electric-field-induced anodic oxidation of aminosilane SAM using an ink-free method. In this versatile method, both "positive" and "negative" type pattern transfers are possible. The chemically converted patterns are induced by localized electrical fields on the microcontacted areas, and the patterning resolution is insensitive to the diffusion of oxidizing agents because of the self-limiting oxidation kinetics, thereby enabling high-resolution, large-scale parallel patterning.     

Directed immobilization of protein-coated nanospheres to nanometer-scale patterns fabricated by electron beam lithography of poly(ethylene glycol) self-assembled monolayers

Controlling the spatial organization of biomolecules on solid supports with high resolution is important for a wide range of scientific and technological problems. Here we report a study of electron beam lithography (EBL) patterning of a self-assembled monolayer (SAM) of the amide-containing poly(ethylene glycol) (PEG) thiol CH(3)O(CH(2)CH(2)O)(17)NHCO(CH(2))(2)SH on Au and demonstrate the patterning of biomolecular features with dimensions approaching 40 nm. The electron dose dependence of feature size and pattern resolution is studied in detail by atomic force microscopy (AFM), which reveals two distinct patterning mechanisms. At low doses, the pattern formation occurs by SAM ablation in a self-developing process where the feature size is directly dose-dependent. At higher doses, electron beam-induced deposition of material, so-called contamination writing, is seen in the ablated areas of the SAM. The balance between these two mechanisms is shown to depend on the geometry of the pattern. The patterned SAMs were backfilled with fluorescent 40-nm spheres coated with NeutrAvidin. These protein-coated spheres adhered to exposed areas in the SAM with high selectivity. This direct writing approach for patterning bioactive surfaces is a fast and efficient way to produce patterns with a resolution approaching that of single proteins.     

Dependence of patterned binary alkanethiolate self-assembled monolayers on "UV-photopatterning" conditions and evolution with time, terminal group, and methylene chain length

We have investigated the mechanism of UV photopatterning of binary alkanethiolate self-assembled monolayers (SAMs) adsorbed on Au(111) using time-of-flight secondary ion mass spectrometry. The SAMs were photopatterned using a 500 W Hg arc lamp. The patterning process is strongly dependent on the wavelength of light used. When an unfiltered arc lamp is employed, IR light impinges on the sample and causes considerable sample heating. Methyl-terminated SAMs with less than 14 carbons in the chain melt at the temperatures reached and become very disordered and so can be easily displaced by a second SAM. This leads to significant pattern degradation ("erosion"). SAMs with greater than 14 carbons undergo a transition to an incommensurate phase but remain stable on the surface, and the pattern is retained. When the IR light is filtered out, a different behavior is observed. UV-photopatterned methyl-terminated SAMs with 10 carbons in the chain are stable. Terminal group interactions, such as H-bonding, provide extra stabilization energy during photopatterning, so some patterns with shorter carbon chains may also be stable. The displacement of the photooxidized SAMs on the patterned surface follows kinetics similar to that of large-area SAM formation.     

A new HPLC–MS/MS method for investigating degradation kinetics of 6‴-feruloylspinosin and identifying its metabolites by rat intestinal bacterial flora in vitro

6?-Feruloylspinosin, a C-glycoside flavonoid, is the active ingredient responsible for the anxiolytic and sedative effects of Zizyphi Spinosiae Semen. However, the degradation mechanism of 6?-feruloylspinosin in vivo is largely unknown. In this study, we developed a simple and rapid method for determining the degradation of 6?-feruloylspinosinin in the bacterial culture using high-performance liquid chromatography (HPLC)–tandem mass spectrometry (MS). The precisions were less than 2.93%, and the accuracy (RE%) ranged from ?4.07 to 3.09%. The HPLC–MS/MS method was successfully applied to study the degradation kinetics of 6?-feruloylspinosin and identify its metabolic derivatives. The results indicated that the degradation kinetics and the rate constants were correlated with the concentration of sample solutions and the degradation process could be classified as apparent first-order kinetic reaction. Obviously, the degradation rate of 6?-feruloylspinosin was rather rapid as 80% of the compound could be decomposed in only 8–12?hr. Furthermore, two metabolites were identified from the serial hydrolysis of 6?-feruloylspinosin.     

Self‐Assembly Film of Zeolite Y Nanocrystals Loading Palladium on an Au Electrode for Electrochemical Applications

Nano‐scale zeolite Y crystals were synthesized, and palladium nanoparticles were prepared in the supercage of the zeolite by “ship‐in‐a‐bottle” approach. A novel method to fabricate zeolite‐modified electrode (ZME) loading Pd nanoparticles was developed, in which the zeolite Y loading Pd²⁺ ions was self‐assembled on (3‐mercaptopropyl) trimethoxysilane‐attached Au surface to form the stable and density packed multilayers (SAM‐ZME). The structures of zeolite Y and the SAM‐ZME were investigated by using TEM, XRD and SEM techniques. Pd²⁺ ions in the SAM‐ZME were converted into Pd nanoparticles (Pd_n⁰) by two steps consisting of the electrochemical reduction as well as the succeeding admission and release of CO. The redox couple [Fe(CN)₆]^3?/4? was used to probe the electron‐transfer barrier properties during self‐assembling process. Moreover, the special properties of the SAM‐ZME loading Pd_n⁰ were studied by using cyclic voltammetry and CO‐probe in situ FTIR spectroscopy. The results illustrated that Pd_n⁰ in the SAM‐ZME exhibits higher electrocatalytic activity for oxidation of adsorbed CO than that of ZME prepared in our previous study by zeolite coating method. The present study is of importance in design and preparation of SAM‐ZME, which poccesseses excellent properties for the immobilization of electrocatalysts or biomolecules.     

Se-Adenosyl-l-selenomethionine Cofactor Analogue as a Reporter of Protein Methylation

Posttranslational methylation by S-adenosyl-l-methionine(SAM)-dependent methyltransferases plays essential roles in modulating protein function in both normal and disease states. As such, there is a growing need to develop chemical reporters to examine the physiological and pathological roles of protein methyltransferases. Several sterically bulky SAM analogues have previously been used to label substrates of specific protein methyltransferases. However, broad application of these compounds has been limited by their general incompatibility with native enzymes. Here we report a SAM surrogate, ProSeAM (propargylic Se-adenosyl-l-selenomethionine), as a reporter of methyltransferases. ProSeAM can be processed by multiple protein methyltransferases for substrate labeling. In contrast, sulfur-based propargylic SAM undergoes rapid decomposition at physiological pH, likely via an allene intermediate. In conjunction with fluorescent/affinity-based azide probes, copper-catalyzed azide-alkyne cycloaddition chemistry, in-gel fluorescence visualization and proteomic analysis, we further demonstrated ProSeAM's utility to profile substrates of endogenous methyltransferases in diverse cellular contexts. These results thus feature ProSeAM as a convenient probe to study the activities of endogenous protein methyltransferases.     

Computer simulation of adhesion between hydrophilic and hydrophobic self-assembled monolayers in water

The grand canonical Monte Carlo technique and atomistic force fields are used to calculate the force-distance relations and free energies of adhesion between carboxyl and methyl terminated alkanethiolate self-assembled monolayers (SAMs) in water. Both symmetric and asymmetric confinements are considered, as formed by like and unlike SAMs, respectively. As the confinement is increased, water confined by the hydrophobic methyl terminated SAMs experiences capillary evaporation. As a consequence, the adhesion energy is determined by the direct interaction between bare SAMs. In the asymmetric system, an incomplete capillary evaporation is observed, with the number of water molecules dropped by more than an order of magnitude. The remaining water molecules are all adsorbed on the hydrophilic SAM, while the hydrophobic SAM is separated from the rest of the system by a thin vapor layer. The calculated free energies of adhesion are in acceptable agreement with experiment.     

Ozonolysis and photolysis of alkene-terminated self-assembled monolayers on quartz nanoparticles: implications for photochemical aging of organic aerosol particles

Photolysis of alkene-terminated self assembled monolayers (SAM) deposited on Degussa SiO(2) nanoparticles is studied following oxidation of SAM with a gaseous ozone/oxygen mixture. Infrared cavity ring-down spectroscopy is used to observe gas-phase products generated during ozonolysis and subsequent photolysis of SAM in real time. Reactions taking place during ozonolysis transform alkene-terminated SAM into a photochemically active state capable of photolysis in the tropospheric actinic window (lambda > 295 nm). Formaldehyde and formic acid are the observed photolysis products. Photodissociation action spectra of oxidized SAM and the observed pattern of gas-phase products are consistent with the well-established Criegee mechanism of ozonolysis of terminal alkenes. There is strong evidence for the presence of secondary ozonides (1,3,4-trioxalones) and other peroxides on the oxidized SAM surface. The data imply that photolysis plays a role in atmospheric aging of primary and secondary organic aerosol particles.     

Fibronectin and proteoglycans as determinants of cell-substratum adhesion.

When normal or SV40-transformed Balb/c 3T3 cells are treated with the Ca++-specific chelator EGTA, they round up and pull away from their footpad adhesion sites to the serum-coated tissue culture substrate, as shown by scanning electron microscope studies. Elastic membranous retraction fibers break upon culture agitation, leaving adhesion sites as substrate-attached material (SAM) (Cells leave "footprints" of substrate adhesion sites during movement by a very similar process.) SAM contains 1-2% of the cell's total protein and phospholipid content and 5-10% of its glucosamine-radiolabeled polysaccharide, most of which is glycosaminoglycan (GAG). By one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis, there is considerable enrichment in SAM for specific GAGs; for the glycoprotein fibronectin; and for the cytoskeletal proteins actin, myosin, and the subunit protein of the 10 nm-diameter filaments. Fibrillar fibronectin of cellular origin and substratum-bound fibronectin of serum origin (cold-insoluble globulin, CIg) have been visualized by immunofluorescence microscopy. The GAG composition in SAM has been examined under different cellular growth and attachment conditions. Heparan sulfate content correlates with glycopeptide content (derived from glycoprotein). Newly attaching cells deposit SAM with principally heparan sulfate and fibronectin and little of the other GAGs. Hyaluronate and chrondroitin proteoglycans are coordinately deposited in SAM as cells begin spreading and movement over the substrate. Cells attaching to serum-coated or CIg-coated substrates deposited SAM with identical compositions. The proteoglycan nature of the GAGs in SAM has been examined, as well as the ability of proteoglycans to form two classes of reversibly dissociable "supramolecular complexes" - one class with heparan sulfate and glycopeptide-containing material and the second with hyaluronate-chondroitin complexes. Enzymatic digestion of "intact" SAM with trypsin or testicular hyaluronidase indicates that (1) only a small portion of long-term radiolabeled fibronectin and cyto-skeletal protein is bound to the substrate via hyaluronate or chondroitin classes of GAG; (2) most of the fibronectin, cytoskeletal protein and heparan sulfate coordinately resist solubilization; and (3) newly synthesized fibronectin, which is metabolically labile in SAM, is linked to SAM by hyaluronate- and/or chondroitin-dependent binding. All of our studies indicate that heparan sulfate is a direct mediator of adhesion of cells to the substrate, possibly by binding to both cell-surface fibronectin and substrate-bound CIg in the serum coating; hyaluronate-chondroitin complexes in SAM appear to be most important in motility of cells by binding and labilizing fibronectin at the periphery of footpad adhesions, with subsequent cytoskeletal disorganization.     

The [4Fe-4S](2+) cluster in reconstituted biotin synthase binds S-adenosyl-L-methionine

The combination of resonance Raman, electron paramagnetic resonance and M?ssbauer spectroscopies has been used to investigate the effect of S-adenosyl-l-methionine (SAM) on the spectroscopic properties of the [4Fe-4S]2+ cluster in biotin synthase. The results indicate that SAM interacts directly at a unique iron site of the [4Fe-4S]2+ cluster in BioB and support the hypothesis of a common inner-sphere mechanism for the reductive cleavage of SAM in the radical SAM family of Fe-S enzymes.     

Analytical curve or standard addition method: how to elect and design--a strategy applied to copper determination in sugarcane spirits using AAS

In most instrumental analysis, the analyte concentration is usually obtained the by Analytical Curve Method (ACM) or Standard Addition Method (SAM). Thus, it is important for the analyst to select the most appropriate method, to seek the best conditions of analysis, and to provide parameters of analytical performance. A strategy to do so is proposed in this paper in conjunction with MATLAB software to implement it. The proposed strategy was applied to copper determination by atomic absorption spectrometry in Brazilian sugarcane spirits termed 'Cacha?a' and SAM was chosen as the most appropriate method. To select the best experimental design for SAM, the influence of some factors, such as the number of standard additions and concentration levels, the location of the levels and the average concentration of the standard additions were demonstrated. The design with six standard additions, four concentration levels located near the inferior and superior levels and the average concentration of the standard additions closer to zero yielded SAM with an adequate compromise between precision, cost and time of analysis. The uniform distribution of concentration levels, usually used in routine analysis, is not a good design regarding precision. On the other hand, it is adequate when the linear range is unknown. Generally, the proposed strategy can be applied to different instrumental techniques and samples, which aim to improve their analytical performance.     

Soft colloidal lithography by strong physical contact using swollen magnetic microspheres and magnetic force

Herein we report on pattern formation of a self-assembled monolayer (SAM) using soft colloidal lithography, based on strong physical contact between microspheres and substrate. In typical colloidal lithography, weak contact of microspheres with the substrate does not block the formation of a SAM. To establish conformal contact between microspheres and substrate to block the formation of a SAM, magnetic force was exerted on softened paramagnetic microspheres that had been swollen by a solvent. The soft colloidal lithography with controlled buoyance enables pattern formation through simple wet chemistry.     

Effect of the electrostatic interaction on the redox reaction of positively charged cytochrome C adsorbed on the negatively charged surfaces of acid-terminated alkanethiol monolayers on a Au(111) electrode

The electrochemical properties of cytochrome c (cyt c) adsorbed on mixed self-assembled monolayers (SAMs) of 2-mercaptoethanesulfonate (MES)/2-mercaptoethanol (MEL) are compared with those on single-component SAMs of MES, MEL, and mercaptopropionic acid (MPA), using cyclic voltammetry and potential-modulated UV-vis reflectance spectroscopy. The rate constant of electron transfer (ET), k(et), of cyt c adsorbed on the SAM of MPA decreases from 1450 +/- 210 s(-1) at pH 7 to 890 +/- 100 s(-1) at pH 9. In contrast, the value of k(et) of cyt c on the SAM of MES is pH-independent at 100 +/- 15 s(-1). Those facts suggest that a large negative charge density on the SAM surface slows down the ET between cyt c and the electrode. The surface charge density of the SAM affects also the amount of electroactive cyt c, Gamma(e), which decreases from 10.0 +/- 1.0 to 5.3 +/- 1.1 pmol cm(-2) with increasing pH from 7 to 9 on the SAM of MPA. Similarly, the k(et) of cyt c adsorbed on the mixed SAMs of MES/MEL sharply decreases from 900 +/- 300 s(-1) to 110 s(-1) as the surface mole fraction of MES increases beyond 0.5, suggesting the presence of a negative surface charge threshold beyond which the rate of ET of cyt c is dramatically lowered. The decrease in the k(et) on the SAMs at high negative charge densities probably results from the confinement of adsorbed cyt c by the strong electrostatic force to an orientation that is not optimal for the ET reaction.     

Identification of degradation impurities in aripiprazole oral solution using LC–MS and development of validated stability indicating method for assay and content of two preservatives by RP-HPLC

A simple and simultaneous reverse phase high-performance liquid chromatographic method was developed for the quantification of aripiprazole (ARI) and two preservatives, namely, methyl paraben and propyl paraben in ARI oral solution. The method was developed on ACE C18 (4.6?×?250?mm, 5?µm) column using gradient elution of 0.1% v/v trifluoroacetic acid in water and acetonitrile as mobile phase components. Flow rate of 1.0?mL/min and 30°C column temperature were used for the method at quantification wavelength of 254?nm. The developed method was validated in accordance with International Conference on Harmonization guideline for various parameters. Forced degradation study was conducted in acid, base, peroxide, heat, and light stress conditions. ARI was found to degrade in oxidation, acid hydrolysis, and heat while it was stable under the remaining conditions. Specificity of the method was verified using Photo Diode Array (PDA) detector by evaluating purity of peaks from degradation samples. Major degradation impurities formed during stress study were identified using liquid chromatography–mass spectrometry method. The present method was useful for determining the content of all the three main analytes present in the oral solution without interference from degradation impurities. The method was robust under the deliberately modified conditions.     

Simultaneous detection of S-adenosylmethionine and S-adenosylhomocysteine in mouse and rat tissues by capillary electrophoresis.

A capillary electrophoresis method for the determination of S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) in rat liver and kidney and mouse liver is described. The method can also be used to determine SAM in whole blood. The method provides rapid (approximately 16 min sample to sample) resolution of both compounds in perchloric extracts of tissues. Separation was performed by using an uncoated 50 microm ID capillary with 60 cm total length (50 cm to the detector window). Samples were separated at 22.5 kV and the separation running buffer was 200 mM glycine pH 1.8 (with HCl). The method compares favorably to HPLC methods (r (2) = 0.994 for SAM, r (2) = 0.998 for SAH) and has a mass detection limit of about 10 fmol for both SAM and SAH at a signal-to-noise ratio of 3. The method is linear over ranges of 1-100 microM SAM and 1-250 microM SAH. This method can be used to determine tissue concentrations of SAM and SAH, two metabolites that can provide insight into many biological processes.     

硅基底金纳米粒子微阵列电极的制备及其电化学性质的研究— 多孔硅电致发光 生物传感器研究的前期探索

用Frens法制备了不同粒径的金纳米粒子,并用透射电镜、紫外可见分光光度法进行了表征。用自组装技术得到了金膜电极表面的金纳米粒子二维阵列电极,用扫描电镜、电化学等方法对该微阵列电极进行了表征。结果表明,当金电极表面被自组装膜完全覆盖后,电化学反应不再发生,而将金纳米粒子组装到膜上以膈,才得到电化学信号。我们认为,金纳米粒子在这里对电荷的跨膜转移有很强的促进作用。对于该过程的研究,用助于理解电荷的转移机制,对进一步理解电荷隧穿过程有一定的指导意义。     

Multifunctional mixed SAMs that promote both cell adhesion and noncovalent DNA immobilization

The ability of DNA strands to influence cellular gene expression directly and to bind with high affinity and specificity to other biological molecules (e.g., proteins and target DNA strands) makes them a potentially attractive component of cell culture substrates. On the basis of the potential importance of immobilized DNA in cell culture and the well-defined characteristics of alkanethiol self-assembled monolayers (SAMs), the current study was designed to create multifunctional SAMs upon which cell adhesion and DNA immobilization can be independently modulated. The approach immobilizes the fibronectin-derived cell adhesion ligand Arg-Gly-Asp-Ser-Pro (RGDSP) using carbodiimide activation chemistry and immobilizes DNA strands on the same surface via cDNA-DNA interactions. The surface density of hexanethiol-terminated DNA strands on alkanethiol monolayers (30.2-69.2 pmol/cm2) was controlled using a backfill method, and specific target DNA binding on cDNA-containing SAMs was regulated by varying the soluble target DNA concentration and buffer characteristics. The fibronectin-derived cell adhesion ligand GGRGDSP was covalently linked to carboxylate groups on DNA-containing SAM substrates, and peptide density was proportional to the amount of carboxylate present during SAM preparation. C166-GFP endothelial cells attached and spread on mixed SAM substrates and cell adhesion and spreading were specifically mediated by the immobilized GGRGDSP peptide. The ability to control the characteristics of noncovalent DNA immobilization and cell adhesion on a cell culture substrate suggests that these mixed SAMs could be a useful platform for studying the interaction between cells and DNA.     

Creation of chiral adsorbed structures using external inputs: results from lattice Monte Carlo simulations

Patterning of solid surfaces with organic molecules has been recognized as a promising method to create functional 2D matrices with tunable structure and properties. In this work we use the lattice Monte Carlo simulations to study chiral pattern formation in adsorbed systems comprising simple molecular building blocks differing in shape. To that end we consider five-membered rigid isomers whose composite segments can occupy vertices of a triangular lattice and interact with short-range (nearest neighbors) forces. Our main focus is on those molecules which are prochiral, that is they can adopt mirror-image planar configurations when adsorbed. Moreover, the effect of orientational in-plane confinement of the molecules, which reflects their coupling with an external directional field, on the structure formation and chiral resolution in 2D is explored. The obtained results demonstrate that the confinement imposed on the surface enantiomers can induce their resolution and formation of extended homochiral domains. However, it is also shown that for certain molecular shapes the confinement can produce mixed racemic crystals instead of the homochiral assemblies. The insights from our simulation studies can be helpful in preliminary screening of molecular libraries to select optimal building blocks able to self-assembly into chiral 2D patterns with predefined architecture.     

High‐Resolution Surface Chemical Analysis of a Trifunctional Pattern Made by Sequential Colloidal Shadowing

We present a new method for creating surface chemical patterns where three chemistries can be periodically arranged at alternate positions on a single substrate without the use of top‐down approaches. High‐resolution chemical imaging by time‐of‐flight secondary ion mass spectrometry (ToF‐SIMS), with nanometer spatial resolution, is used to prove the success of the patterning and subsequent chemical modification steps. We use a combination of colloidal self‐assembly, plasma etching, self‐assembled monolayers (SAMs) and physical vapour deposition (PVD). The method utilizes a double colloid assembly process in which a first layer of close‐packed colloids is created, followed by plasma etching, coating with gold and deposition of a first SAM layer. A second particle layer is deposited on top of the first layer masking the interstitial spaces containing the first SAM. A second gold layer is deposited followed by a second SAM. After particle removal the surface consists of the pattern containing two different SAMs and a SiO₂ layer that can be readily functionalized with silanes. The possibility in the replacement of the two different thiols is investigated by X‐ray photoelectron spectroscopy (XPS) and it was found that no replacement is taking place. ToF‐SIMS imaging is used to show the periodicity of the chemical patterns by tracking unique fragment ions from the different surface regions. The patterning method is adaptable to create smaller or larger chemical patterns by appropriate choice of particle sizes. The patterns are useful for immobilizing biomolecules for cell studies or as multiplexed biosensors.