Functionalized surface arrays for spatial targeting of immune cell signaling

The effect of surface topography and chemistry on cellular response is of fundamental importance, especially where living systems encounter device surfaces as in medical implants, tissue engineering, and cell-based sensors. To understand these biological processes on surfaces, there is a widespread interest in tailored surface-active materials produced by a combination of surface chemistry coupled to advanced patterning processes. We utilize self-assembled monolayers (SAMs) as molecular templates with submicrometer-scale spatial resolution to engage and cluster IgE receptors on rat basophilic leukemia (RBL) mast cells. Bioactive templates consisted of gold arrays on silicon with patterns from 1 mum down to 45 nm. These gold arrays served as molecular tethering sites, enabling covalent binding of functionalized self-assembled monolayers of alkanethiols. The free ends of the monolayers were functionalized with 2,4-dinitrophenyl(DNP)-caproate-based ligands which interact specifically with anti-DNP IgE bound to its high affinity cell surface receptor, FcepsilonRI on RBL mast cells. Present results on structures 1 mum down to 600 nm in size indicate that these ligand-immobilized patterned arrays can function as a powerful tool for visualization and systematic characterization of cell membrane involvement in IgE receptor-mediated immune cell signaling.     

Alkanephosphonates on hafnium-modified gold: a new class of self-assembled organic monolayers

A new method for assembling organic monolayers on gold is reported that employs hafnium ions as linkers between a phosphonate headgroup and the gold surface. Monolayers of octadecylphosphonic acid (ODPA) formed on gold substrates that had been pretreated with hafnium oxychloride are representative of this new class of organic thin films. The monolayers are dense enough to completely block assembly of alkanethiols and resist displacement by alkanethiols. The composition and structure of the monolayers were investigated by contact angle goniometry, XPS, PM-IRRAS, and TOF-SIMS. From these studies, it was determined that this assembly strategy leads to the formation of ODPA monolayers similar in quality to those typically formed on metal oxide substrates. The assembly method allows for the ready generation of patterned surfaces that can be easily prepared by first patterning hafnium on the gold surface followed by alkanephosphonate assembly. Using the bifunctional (thiol-phosphonate) 2-mercaptoethylphosphonic acid (2-MEPA), we show that this new assembly chemistry is compatible with gold-thiol chemistry and use TOF-SIMS to show that the molecule attaches through the phosphonate functionality in the patterned region and through the thiol in the bare gold regions. These results demonstrate the possibility of functionalizing metal substrates with monolayers typically formed on metal oxide surfaces and show that hafnium-gold chemistry is complementary and orthogonal to well-established gold-thiol assembly strategies.     

Expedient generation of patterned surface aldehydes by microfluidic oxidation for chemoselective immobilization of ligands and cells

An expedient and inexpensive method to generate patterned aldehydes on self-assembled monolayers (SAMs) of alkanethiolates on gold with control of density for subsequent chemoselective immobilization from commercially available starting materials has been developed. Utilizing microfluidic cassettes, primary alcohol oxidation of tetra(ethylene glycol) undecane thiol and 11-mercapto-1-undecanol SAMs was performed directly on the surface generating patterned aldehyde groups with pyridinium chlorochromate. The precise density of surface aldehydes generated can be controlled and characterized by electrochemistry. For biological applications, fibroblast cells were seeded on patterned surfaces presenting biospecifc cell adhesive (Arg-Glyc-Asp) RGD peptides.     

Using self-polymerized dopamine to modify the antifouling property of oligo(ethylene glycol) self-assembled monolayers and its application in cell patterning

We report a one-step, mild method to modify antifouling oligo(ethylene glycol)-terminated self-assembled monolayers. We demonstrate for the first time that self-polymerized dopamine, previously reported as an underwater adhesive, can be patterned on typical antifouling surfaces by microfluidic patterning or microcontact printing. The patterns can be applied in spatiotemporal cell patterning.     

Local control of protein binding and cell adhesion by patterned organic thin films

Control of the cell adhesion and growth on chemically patterned surfaces is important in an increasing number of applications in biotechnology and medicine, for example implants, in-vitro cellular assays, and biochips. This review covers patterning techniques for organic thin films suitable for site-directed guidance of cell adhesion to surfaces. Available surface patterning techniques are critically evaluated, with special emphasis on surface chemistry that can be switched in time and space during cultivation of cells. Examples from the authors’ laboratory include the use of cell-repellent self-assembled monolayers (SAM) terminated by oligoethylene glycol (OEG) units and the lifting of the cell repellent properties by use of electrogenerated Br₂/HOBr which can be performed with positionable microelectrodes. Structural changes of the SAM were analyzed by polarization-modulated infrared reflection absorption spectroscopy (PM IRRAS). Use of a soft array system of individually addressable microelectrodes enables formation of flexible and complex patterns in a short time and has the potential for further acceleration of probe-induced local manipulation of cell adhesion.     

Template-directed adsorption of block copolymers on alkanethiol-patterned gold surfaces

Functionalized alkanethiols have been self-assembled on gold to modify the wetting properties of the surface and promote or hinder the adsorption of block copolymers containing both hydrophobic and hydrophilic blocks. X-ray photoelectron spectroscopy (XPS) studies of spin-coated polyethylene-block-poly(ethylene oxide) (PE-b-PEO) copolymers on 16-mercaptohexadecanoic acid (MHDA)-, octadecanethiol (ODT)-, and 1H,1H,2H,2H-perfluorodecanethiol (PFDT)-covered surfaces have been performed. In the case of an 80 wt % PEO block copolymer, spin-coating on a gold surface precovered with MHDA results in a polymer film thick enough to completely attenuate Au 4f photoelectrons; spin-coating on the more hydrophobic ODT and PFDT monolayers leads to significantly thinner polymer films and incomplete attenuation of the gold photoelectrons. The opposite results are observed when a 20 wt % PEO block copolymer is used. Angle-resolved XPS studies of the 80 wt % PEO block copolymer spin-coated onto an MHDA-covered surface indicate that the PE blocks of the polymer segregate to the near-surface region, oriented away from the hydrophilic carboxylic acid tails of the monolayers; the surface concentration of PE is further enhanced by annealing at 90 degrees C. Microcontact printing and dip-pen nanolithography have been used to pattern gold surfaces with MHDA, and the surfaces have been backfilled with ODT or PFDT, such that the unpatterned regions of the surface are covered with hydrophobic monolayers. In the case of backfilling with PFDT, spin-coating the 80 wt % PEO copolymer onto these patterned surfaces and subsequent annealing results in the block copolymer preferentially adsorbing on the MHDA-covered regions and forming well-defined patterns that mimic the MHDA pattern, as determined by scanning electron microscopy and atomic force microscopy. Significantly worse patterning, characterized by micron-sized polymer droplets, results when the surface is backfilled with ODT instead of PFDT. Using PFDT and MHDA, polymer features having widths as small as 500 nm have been formed. These studies demonstrate a novel method to pattern block copolymers with nanoscale resolution.     

Microstructuring of solid-supported lipid layers using SAM pattern generation by scanning electrochemical microscopy and the chemical lens

To prepare patterns of adsorption sites for alkanethiols with high lateral resolution, we used the scanning electrochemical microscopy (SECM) to etch masks into uniform layers of nickel coated on gold surfaces. The patterning of the nickel mask was accomplished in aqueous solutions by electrogenerating nitric acid out of nitrite at an ultramicroelectrode. Due to the sluggish kinetics of nickel etching in acidic media, the pattern generated by a 10-microm tip was about 50-microm wide, depending on the duration of the etching. As an alternative, applying the principle of the chemical lens by adding potassium hydroxide as a scavenger, the size of the adsorption sites had been reduced to 4 microm, independent of the duration of etching. In a follow-up step, monolayers of 11-mercaptoundecanoic acid were formed on the exposed gold areas of the surface by self-assembly. Fluorescent liposomes containing tetramethylrhodamine-labeled phospholipids were used to create solid-supported lipid layers (SSLLs). These fluorescent liposomes showed a selective binding affinity to the self-assembled monolayers (SAMs) modified areas, but not to the nickel surface. The patterns generated were imaged by the SECM itself, as well as by optical and fluorescence microscopy.     

Nanometer-scale patterned surfaces for control of cell adhesion.

A novel cell-adhesion surface, controlled by nanometer-scale topography and chemical patterning, was developed using semiconductor fabrication methods and the formation of self-assembled monolayers. The patterned surface had a sharp contrast between the adsorption and non-adsorption of proteins and cells, and the contrast could be maintained for more than 10 days. The patterning method could easily realize a single cell array and control of the cell morphology. The nanometer-scale patterned surface could control cell adhesion and proliferation. Using the patterned surface will contribute to studies about cell-surface interactions.     

Anti-fouling chemistry of chiral monolayers: enhancing biofilm resistance on racemic surface

This work reports the resistance to protein adsorption and bacterial biofilm formation by chiral monolayers of polyol-terminated alkanethiols surrounding micrometer-sized patterns of methyl-terminated alkanethiols on gold films. We discover that patterned surfaces surrounded by chiral polyol monolayers can distinguish different stages of biofilm formation. After inoculation on the surfaces, bacteria first reversibly attached on the chiral polyol monolayers. Over time, the bacteria detached from the polyol surfaces, and attached on the hydrophobic micropatterns to form biofilms. Interestingly, while both enantiomers of gulitol- and mannonamide-terminated monolayer resisted adsorption of proteins (bovine serum albumin, lysozyme, and fibrinogen) and confined biofilms formed on the micropatterns, the monolayers formed by the racemic mixture of either pair of enantiomers exhibited stronger antifouling chemistry against both protein adsorption and biofilm formation than monolayers formed by one enantiomer alone. These results reveal the different chemistries that separate the different stages of biofilm formation, and the stereochemical influence on resisting biofoulings at a molecular-level.     

Effect of surface composition on the adsorption of photosystem I onto alkanethiolate self-assembled monolayers on gold

We have used self-assembled monolayers (SAMs) prepared from omega-terminated alkanethiols on gold to generate model surfaces and examine the effect of surface composition on the adsorption of Photosystem I (PSI), stabilized in aqueous solution by Triton X-100. Triton-stabilized PSI adsorbs to high-energy surfaces prepared from HO- and HO2C-terminated alkanethiols but does not adsorb to low-energy surfaces. The inhibition of PSI adsorption at low-energy surfaces is consistent with the presence of a layer of Triton X-100 that adsorbs atop the hydrophobic SAM and presents a protein-resistant poly(ethylene glycol) (PEG) surface. While the presence of the PEG surface prevents the adsorption of PSI, the displacement of the inhibiting layer of Triton X-100 by dodecanol, a more active surfactant, greatly enhances the adsorption of PSI. This inhibiting effect by Triton X-100 can be extended to other protein systems such as bovine serum albumin.     

Reversible covalent patterning of self-assembled monolayers on gold and silicon oxide surfaces

This paper describes the generation of reversible patterns of self-assembled monolayers (SAMs) on gold and silicon oxide surfaces via the formation of reversible covalent bonds. The reactions of (patterned) SAMs of 11-amino-1-undecanethiol (11-AUT) with propanal, pentanal, decanal, or terephthaldialdehyde result in dense imine monolayers. The regeneration of these imine monolayers to the 11-AUT monolayer is obtained by hydrolysis at pH 3. The (patterned) monolayers were characterized by Fourier transform infrared reflection absorption spectroscopy, X-ray photoelectron spectroscopy, contact angle and electrochemical measurements, and atomic force microscopy. Imines can also be formed by microcontact printing of amines on terephthaldialdehyde-terminated substrates. Lucifer Yellow ethylenediamine was employed as a fluorescent amine-containing marker to visualize the reversible covalent patterning on a terephthaldialdehyde-terminated glass surface by confocal microscopy. These experiments demonstrate that with reversible covalent chemistry it is possible to print and erase chemical patterns on surfaces repeatedly.     

Adsorption of ethoxylated cationic surfactants on self-assembled monolayers of alkanethiols on gold using surface plasmon resonance detection

Adsorption of a series of ethoxylated cationic surfactants at model surfaces of alkanethiol self-assembled monolayers was studied by the surface plasmon resonance technique. Model surfaces were tailor-made by choosing alkanethiols or mixtures of alkanethiols with methyl, hydroxyl, carboxyl, and trimethylammonium groups in terminal position. The ethoxylated and quaternized cationic surfactants having from 2 to 18 oxyethylene units, showed a decrease in adsorbed amount with increasing oxyethylene chain length for both hydrophobic and hydrophilic surfaces. On a negatively charged surface, containing carboxylate groups, the surfactant with only two oxyethylene groups adsorbed strongly due to electrostatic attraction and the adsorption increased with increasing amount of surface carboxylate groups. This work shows the usefulness of self-assembled alkanethiols on gold as a tool for performing surfactant adsorption studies on surfaces with variable hydrophobicity and charge.     

Blocking behavior of self-assembled monolayers on gold electrodes

Self-assembled monolayers (SAMs) with metal electrodes, especially thiols on gold, are the subject of this investigation because of the unique properties of SAM-modified surfaces. Normal alkanethiols are used to modify the surface of a conventional gold electrode to block certain ions such as Pb(II) and Cu(II) from the surface of the electrode. Normal alkanethiols are also used to study the SAM-gold interfacial adsorption-desorption behavior of the self-assembled monolayer. The effects of varying chain length of SAMs, varying concentration of the alkanethiol solutions, immersion time of the pure gold electrode in the SAM solution, and the stability of a SAM-modified gold electrode in fresh chloroform are investigated using the oxidation-reduction peaks of gold. Conditions that optimize the surface coverage and the uniformity of the SAMs have been determined. Normal alkanethiols proved to be a good insulator on the electrode surface. Received: 16 January 1997 / Accepted: 4 March 1997     

Ultra-rapid laser protein micropatterning: screening for directed polarization of single neurons

Protein micropatterning is a powerful tool for studying the effects of extracellular signals on cell development and regeneration. Laser micropatterning of proteins is the most flexible method for patterning many different geometries, protein densities, and concentration gradients. Despite these advantages, laser micropatterning remains prohibitively slow for most applications. Here, we take advantage of the rapid multi-photon induced photobleaching of fluorophores to generate sub-micron resolution patterns of full-length proteins on polymer monolayers, with sub-microsecond exposure times, i.e. one to five orders of magnitude faster than all previous laser micropatterning methods. We screened a range of different PEG monolayer coupling chemistries, chain-lengths and functional caps, and found that long-chain acrylated PEG monolayers are effective at resisting non-specific protein adhesion, while permitting efficient cross-linking of biotin-4-fluorescein to the PEG monolayers upon exposure to femtosecond laser pulses. We find evidence that the dominant photopatterning chemistry switches from a two-photon process to three- and four-photon absorption processes as the laser intensity increases, generating increasingly volatile excited triplet-state fluorophores, leading to faster patterning. Using this technology, we were able to generate over a hundred thousand protein patterns with varying geometries and protein densities to direct the polarization of hippocampal neurons with single-cell precision. We found that certain arrays of patterned triangles as small as neurite growth cones can direct polarization by impeding the elongation of reverse-projecting neurites, while permitting elongation of forward-projecting neurites. The ability to rapidly generate and screen such protein micropatterns can enable discovery of conditions necessary to create in vitro neural networks with single-neuron precision for basic discovery, drug screening, as well as for tissue scaffolding in therapeutics.     

Local desorption of thiols by scanning electrochemical microscopy: patterning and tuning the reactivity of self-assembled monolayers

Self-assembled monolayers (SAMs) are widely used in the field of nanotechnologies and (bio)sensors. The monolayer surface properties are tailored by employing several techniques. A large set of SAM post-modification routes are commonly performed to adapt them to a variety of nano-technological and bio-technological studies as well as to several bio-sensoristic applications. Here, we report a procedure to locally modify SAMs by electrochemical desorption of alkanethiols in order to create microsized spots of bare gold area without affecting the surrounding monolayer stability. The tip of the scanning electrochemical microscope (SECM) was employed to draw microstructured pattern according to a defined geometry. The time stability of the pattern was also tested. Furthermore, the patterned surface was post-functionalized using the same alkanethiol or a ferrocene-terminated thiol, in order to tune the surface reactivity of the microstructure. The local surface properties, including reactivity and electron transfer kinetics toward redox mediator reduction, were characterized by SECM.     

An interfacial oxime reaction to immobilize ligands and cells in patterns and gradients to photoactive surfaces

We report a molecularly controlled interfacial chemoselective methodology to immobilize ligands and cells in patterns and gradients to self-assembled monolayers on gold. This strategy is based on reacting soluble ketone or aldehyde tethered ligands to surface-bound oxyamine alkeanethiols to generate a covalent oxime linkage to the surface. We characterize the kinetic behavior of the reaction on the surface with ferrocenecarboxaldehyde (FcCHO) as a model ligand. The precise extent of immobilization and therefore surface density of FcCHO on the SAM is monitored and determined by cyclic voltammetry, which shows a peudo-first-order rate constant of 0.13 min(-1). In order to generate complex surface patterns and gradients of ligands on the surface, we photoprotected the oxyamine group with nitroveratryloxycarbonyl (NVOC). We show that ultraviolet light irradiation through a patterned microfiche film reveals the oxyamine group and we characterize the rate of deprotection by immobilization of ketone containing redox active groups. Finally, we extend this strategy to show biospecific cell attachment of fibroblast cells by immobilizing ketone-GRGDS peptides in patterns. The interfacial oxime reaction is chemoselective and stable at physiological conditions (pH 7.0, 37 degrees C) and may potentially be used to install ligands on the surface in the presence of attached cells to modulate the cell microenvironment to generate dynamic surfaces for monitoring changes in cell behavior in real time.     

Stereochemical effects of chiral monolayers on enhancing the resistance to mammalian cell adhesion

This work describes the different durations of surface confinement of adhered mammalian cells by monolayers comprised of enantiomers of bio-inert polyol-terminated alkanethiols. Enhanced resistance to protein adsorption and cell adhesion is obtained on monolayers formed by a racemic mixture of the enantiomeric alkanethiols.     

Developing a self-assembled monolayer microarray to study stem cell differentiation

We have developed a novel strategy to generate self-assembled monolayer microarray (SAMs-Array) of alkanethiolates on gold surfaces for the study of human mesenchymal stem cells (hMSCs) differentiation. Electroactive alkanethiols were microarray-printed in varying densities to probe the chemical effects on stem cell differentiation. Cyclic voltammetry (CV) was used for the precise determination of the amount of alkanethiol molecules transferred and SAMs formed on the microarray. We can also control the oxidative and reductive state of each molecule displayed to cells by CV. Based on this SAMs-Array technology, we generated a platform for potential high-throughput screening of various surface chemistry effects on cell behaviors for future applications in biomaterials and tissue engineering.     

Plasma stencilling methods for cell patterning

In this paper we describe plasma stencilling techniques for patterning 10 mammalian cell lines on hydrophobic and cell repellent poly(dimethylsiloxane) (PDMS), methylated glass and bacterial grade polystyrene surfaces. An air plasma produced with a Tesla generator operating at atmospheric pressure was used with microengineered stencils for patterned surface oxidation, selectively transforming the surface to a hydrophilic state to enable cell adhesion and growth. Plasma stencilling obviates the need for directly patterning cell adhesion molecules. Instead, during cell culture, adhesion proteins from the media assemble in a bioactive form on the hydrophilic regions. Critically, the removal of protein patterning prior to cell culture provides the option to also use PDMS–PDMS plasma bonding to incorporate cell patterns within microfluidic systems. Linear patterns were generated using PDMS microchannel stencils, and polyimide stencils with through holes were used for the production of cellular arrays. For the production of smaller cellular arrays, a novel microcapillary-based dielectric barrier discharge system was developed. A numerical method to characterise the cell patterns is also introduced and was used to demonstrate that plasma stencilling is highly effective, with complete patterns confined during long term cell culture (>10 days). In summary, plasma stencilling is simple, rapid, inexpensive, reproducible and a potentially universal cell line patterning capability.

Controlling film morphology in conjugated polymer:fullerene blends with surface patterning

We study the effects of patterned surface chemistry on the microscale and nanoscale morphology of solution-processed donor/acceptor polymer-blend films. Focusing on combinations of interest in polymer solar cells, we demonstrate that patterned surface chemistry can be used to tailor the film morphology of blends of semiconducting polymers such as poly-[2-(3,7-dimethyloctyloxy)-5-methoxy-p-phenylenevinylene] (MDMO-PPV), poly-3-hexylthiophene (P3HT), poly[(9,9-dioctylflorenyl-2,7-diyl)-co-benzothiadiazole)] (F8BT), and poly(9,9-dioctylfluorene-co-bis-N,N'-(4-butylphenyl)-bis-N,N'-phenyl-1,4-phenylendiamine) (PFB) with the fullerene derivative, [6,6]-phenyl-C(61)-butyric acid methyl ester (PCBM). We present a method for generating patterned, fullerene-terminated monolayers on gold surfaces and use microcontact printing and Dip-Pen Nanolithography (DPN) to pattern alkanethiols with both micro- and nanoscale features. After patterning with fullerenes and other functional groups, we backfill the rest of the surface with a variety of thiols to prepare substrates with periodic variations in surface chemistry. Spin coating polymer:PCBM films onto these substrates, followed by thermal annealing under nitrogen, leads to the formation of structured polymer films. We characterize these films with Atomic Force Microscopy (AFM), Raman spectroscopy, and fluorescence microscopy. The surface patterns are effective in guiding phase separation in all of the polymer:PCBM systems investigated and lead to a rich variety of film morphologies that are inaccessible with unpatterned substrates. We demonstrate our ability to guide pattern formation in films thick enough to be of interest for actual device applications (up to 200 nm in thickness) using feature sizes as small as 100 nm. Finally, we show that the surface chemistry can lead to variations in film morphology on length scales significantly smaller than those used in generating the original surface patterns. The variety of behaviors observed and the wide range of control over polymer morphology achieved at a variety of different length scales have important implications for the development of bulk heterojunction solar cells.