Micro/nanopatterning of proteins via contact printing using high aspect ratio PMMA stamps and nanoimprint apparatus

Micro- and nanoscale protein patterns have been produced via a new contact printing method using a nanoimprint lithography apparatus. The main novelty of the technique is the use of poly(methyl methacrylate) (PMMA) instead of the commonly used poly(dimethylsiloxane) (PDMS) stamps. This avoids printing problems due to roof collapse, which limits the usable aspect ratio in microcontact printing to 10:1. The rigidity of the PMMA allows protein patterning using stamps with very high aspect ratios, up to 300 in this case. Conformal contact between the stamp and the substrate is achieved because of the homogeneous pressure applied via the nanoimprint lithography instrument, and it has allowed us to print lines of protein approximately 150 nm wide, at a 400 nm period. This technique, therefore, provides an excellent method for the direct printing of high-density sub-micrometer scale patterns, or, alternatively, micro-/nanopatterns spaced at large distances. The controlled production of these protein patterns is a key factor in biomedical applications such as cell-surface interaction experiments and tissue engineering.     

Nanoscopic Pd line arrays using nanocontact printed dendrimers

High-density Pd line arrays with 55 nm line-width were obtained using nanocontact-printed dendrimer monolayers. Elastomeric PDMS stamps for nanocontact printing were replicated from silicon master molds which were fabricated by UV nanoimprinting in combination with reactive ion etching. The fabrication method effectively controlled the aspect ratios of high-density lines for resolving the problems encountered in both replicating silicon masters to PDMS stamps and printing with the replicated PDMS stamps. Using the PDMS nanostamp with an optimized aspect ratio, a self-assembled monolayer of dendrimer was patterned on a Pd film via nanocontact printing, which was facilitated by the strong interaction between Pd and amine groups of the dendrimer. The patterned self-assembled monolayer was used as an etch-resist mask against the wet etchant of Pd, leaving behind a high-density Pd line array over large areas. The resulting functional Pd nanopattern is of practical significance in microelectronics and bio- or gas-sensing devices.     

Microcontact printing of proteins inside microstructures

Microfluidic devices are well suited for the miniaturization of biological assays, in particular when only small volumes of samples and reagents are available, short time to results is desirable, and multiple analytes are to be detected. Microfluidic networks (MFNs), which fill by means of capillary forces, have already been used to detect important biological analytes with high sensitivity and in a combinatorial fashion. These MFNs were coated with Au, onto which a hydrophilic, protein-repellent monolayer of thiolated poly(ethyleneglycol) (HS-PEG) was self-assembled, and the binding sites for analytes were present on a poly(dimethylsiloxane) (PDMS) sealing cover. We report here a set of simple methods to extend previous work on MFNs by integrating binding sites for analytes inside the microstructures of MFNs using microcontact printing (muCP). First, fluorescently labeled antibodies (Abs) were microcontact-printed from stamps onto planar model surfaces such as glass, Si, Si/SiO2, Au, and Au derivatized with HS-PEG to investigate how much candidate materials for MFNs would quench the fluorescence of printed, labeled Abs. Au coated with HS-PEG led to a fluorescence signal that was approximately 65% weaker than that of glass but provided a convenient surface for printing Abs and for rendering the microstructures of the MFNs wettable. Then, proteins were inked from solution onto the surface of PDMS (Sylgard 184) stamps having continuous or discontinuous micropatterns or locally inked onto planar stamps to investigate how the aspect ratio (depth:width) of microstructures and the printing conditions affected the transfer of protein and the accuracy of the resulting patterns. By applying a controlled pressure to the back of the stamp, Abs were accurately microcontact-printed into the recessed regions of MFNs if the aspect ratio of the MFN microstructures was lower than approximately 1:6. Finally, the realization of a simple assay between Abs (used as antigens) microcontact-printed in microchannels and Abs from solution suggests that this method could become useful to pattern proteins in microstructures for advanced bioanalytical purposes.     

Hydrophilic elastomers for microcontact printing of polar inks

A moderately hydrophilic, thermoplastic elastomer (poly(ether-ester)) was investigated as a stamp material for microcontact printing of a polar ink: pentaerythritol-tetrakis-(3-mercaptopropionate). Stamps with a relief structure were produced from this polymer by hot embossing, and a comparison was made with conventional poly(dimethylsiloxane) (PDMS) and oxygen-plasma-treated PDMS. It is shown that the hydrophilic stamps can be used for the repetitive printing (without re-inking) of at least 10 consecutive patterns, which preserve their etch resistance, and this in rather sharp contrast to conventional and oxygen plasma-treated PDMS stamps. It is argued that these enhanced printing characteristics of the hydrophilic stamps originate from an improved wetting and solubility of polar inks in the hydrophilic stamp.     

Self-supporting hydrogel stamps for the microcontact printing of proteins

In this work we explore a new hydrogel stamp material obtained from polymerizing 2-hydroxyethyl acrylate and poly(ethylene glycol) diacrylate in the presence of water for the microcontact printing of proteins directly on gold substrates and by covalent coupling to self-assembled monolayers of alkanethiols. At high cross-link density, the hydrogel is rigid, hydrophilic, and with a high buffer holding capacity to enable the unsupported printing of protein patterns homogeneously and reproducibly, with micrometer-range precision. The stamps were used to print antibodies to human parathyroid hormone, which were shown using immunoassay tests to retain their biological function with binding capacities comparable to those of solution-adsorbed antibodies.     

Bifunctional, chemically patterned flat stamps for microcontact printing of polar inks

Different methods to create chemically patterned, flat PDMS stamps with two different chemical functionalities were compared. The best method for making such stamps, functionalized with 1H,1H,2H,2H-perfluorodecyltrichlorosilane (PFDTS) and 3-(aminopropyl)triethoxysilane (APTS), appeared to be full functionalization of a freshly oxidized flat PDMS stamp with either adsorbate, followed by renewed oxidation through a mask and attachment of the other adsorbate. These stamps were used to transfer polar inks (a thioether-functionalized dendrimer and a fluorescent dye) by microcontact printing. The PFDTS monolayer was used as a barrier against ink transfer, while the APTS SAM areas functioned as an ink reservoir for polar inks. The printing results confirmed the excellent transfer of hydrophilic inks with these stamps to gold and glass substrates, even from aqueous solutions. Attachment of a fluorescent dye on the amino-functionalized regions shows the possibility of the further modification of the chemically patterned stamps for tailoring of the stamps' properties.     

Fishing DNA targets in DNA solutions by using affinity microcontact printing

In this paper, we report the application of affinity microcontact printing (αCP) for "fishing" DNA targets in aqueous solutions and transferring them to solid surfaces for detection purposes. Affinity stamps used in this experiment were made of poly(dimethylsiloxane) (PDMS) with DNA probes covalently immobilized on their surface. When these stamps were immersed in DNA solutions, DNA targets with a perfect-match (PM) sequence to the probes can selectively hybridize to the stamp surfaces and then be transferred to solid surfaces. However, to distinguish PM DNA from single base-pair mismatch (1MM) DNA targets, 10 mM of NaCl must be added to the hybridization buffer. Under the optimized conditions, this αCP can lead to a surface density of PM which is 15 times higher than that of 1MM. The affinity stamp is also able to "fish" PM DNA targets from a mixture of PM/1MM DNA targets and transfer them to solid surfaces. Because DNA probes and targets are separated after printing, we also applied this technique for label-free detection of DNA targets by using liquid crystals.     

Characterization of supported membranes on topographically patterned polymeric elastomers and their applications to microcontact printing

This article describes the fluorescence microscopy and imaging ellipsometry-based characterization of supported phospholipid bilayer formation on elastomeric substrates and its application in microcontact printing of spatially patterned phospholipid bilayers. Elastomeric stamps, displaying a uniformly spaced array of square wells (20, 50, and 100 mum linear dimensions), are prepared using poly(dimethyl)siloxane from photolithographically derived silicon masters. Exposing elastomeric stamps, following UV/ozone-induced oxidation, to a solution of small unilamellar phospholipid vesicles results in the formation of a 2D contiguous, fluid phospholipid bilayers. The bilayer covers both the elevated and depressed regions of the stamp and exhibits a lateral connectivity allowing molecular transport across the topographic boundaries. Applications of these bilayer-coated elastomeric stamps in microcontact printing of lipid bilayers reveal a fluid-tearing process wherein the bilayer in contact regions selectively transfers with 75-90% efficiency, leaving behind unperturbed patches in the depressed regions of the stamp. Next, using cholera-toxin binding fluid POPC bilayers that have been asymmetrically doped with ganglioside Gm1 ligand in the outer leaflets, we examine whether the microcontact transfer of bilayers results in the inversion of the lipid leaflets. Our results suggest a complex transfer process involving at least partial bilayer reorganization and molecular re-equilibration during (or upon) substrate transfer. Taken together, the study sheds light on the structuring of lipid inks on PDMS elastomers and provides clues regarding the mechanism of bilayer transfer. It further highlights some important differences in stamping fluid bilayers from the more routine applications of stamping in the creation of patterned self-assembled monolayers.     

Single etch patterning of stacked silver and molybdenum alloy layers on glass using microcontact wave printing

Stacked thin layers of silver alloy (AgPdCu) and MoCr layers on 10 x 15 cm2 glass substrates were patterned by microcontact wave printing and etching. Patterns of etch-resistant octadecanethiol self-assembled monolayers (SAMs) were wave printed with regular backplane stabilized PDMS stamps. Pattern development was achieved by etching both metal layers in a single step, employing a nitric acid-based etching bath. Trifluoroacetic acid and a nitrite salt were identified as essential bath components for a homogeneous etching process. Etch defects could be eliminated by the addition of a decanesulfonate, which stabilizes the SAM resist via a defect healing mechanism.     

Dendrimer-mediated transfer printing of DNA and RNA microarrays

This paper describes a new method to replicate DNA and RNA microarrays. The technique, which facilitates positioning of DNA and RNA with submicron edge resolution by microcontact printing (muCP), is based on the modification of poly(dimethylsiloxane) (PDMS) stamps with dendrimers ("dendri-stamps"). The modification of PDMS stamps with generation 5 poly(propylene imine) dendrimers (G5-PPI) gives a high density of positive charge on the stamp surface that can attract negatively charged oligonucleotides in a "layer-by-layer" arrangement. DNA as well as RNA is transfer printed from the stamp to a target surface. Imine chemistry is applied to immobilize amino-modified DNA and RNA molecules to an aldehyde-terminated substrate. The labile imine bond is reduced to a stable secondary amine bond, forming a robust connection between the polynucleotide strand and the solid support. Microcontact printed oligonucleotides are distributed homogeneously within the patterned area and available for hybridization. By using a robotic spotting system, an array of hundreds of oligonucleotide spots is deposited on the surface of a flat, dendrimer-modified stamp that is subsequently used for repeated replication of the entire microarray by microcontact printing. The printed microarrays are characterized by homogeneous probe density and regular spot morphology.     

Effect of stamp deformation on the quality of microcontact printing: theory and experiment

Microcontact printing (microCP) is an effective way to generate micrometer- or submicrometer-sized patterns on a variety of substrates. However, the fidelity of the final pattern depends critically on the coupled phenomena of stamp deformation, fluid transfer between surfaces, and the ability of the ink to self-assemble on the substrate. In particular, stamp deformation can produce undesirable effects that limit the practice and precision of microCP. Experimental observations and comparison with theoretical predictions are presented here for three of the most undesirable consequences of stamp deformation: (1) roof collapse of low aspect ratio recesses, (2) buckling of high aspect ratio plates, and (3) lateral sticking of high aspect ratio plates. Stamp behavior was observed visually with an inverted optical microscope while load-displacement data were collected during compression and retraction of stamps. Additionally, a "robotic stamper" was used to deliver ink patterns in precise locations on substrates. These monomolecular ink patterns were then observed in high contrast using the surface potential scanning mode of an atomic force microscope. Theoretical models based on continuum mechanics were used to accurately predict both physical deformation of the stamp and the resultant inking patterns. The close agreement between these models and the experimental data presented clearly demonstrates the essential considerations one must weigh when designing stamp geometry, material, and loading conditions for optimal pattern fidelity.     

Chemically patterned flat stamps for microcontact printing

Locally oxidized patterns on flat poly(dimethylsiloxane) stamps for microcontact printing were used as a platform for the transfer of a hydrophilic fluorescent ink to a glass substrate. The contrast was found to be limited. These locally oxidized patterns were conversely used as barriers for the transfer of hydrophobic n-octadecanethiol. In this case a good contrast was obtained, but the pattern was found to be susceptible to defects (cracks) in the barrier layer. Local stamp surface oxidation and subsequent modification with 1H,1H,2H,2H-perfluorodecyltrichlorosilane, for use as a barrier in the transfer of n-octadecanethiol, 16-mercaptohexadecanoic acid, and octanethiol, resulted in remarkably good contrast and stable patterns. The improved ink transfer control is ascribed to the reduction of undesired surface spreading and a superior mechanical stability of the stamp pattern. This new approach substantially expands the applicability of microcontact printing and provides a tool for the faithful reproduction of even extremely low filling ratio patterns.     

Surface modification of elastomeric stamps for microcontact printing of polar inks

Chemical modification of the surface of a stamp used for microcontact printing (microCP) is interesting for controling the surface properties, such as the hydrophilicity. To print polar inks, plasma polymerization of allylamine (PPAA) was employed to render the surface of poly(dimethylsiloxane) (PDMS), polyolefin plastomers (POP), and Kraton elatomeric stamps hydrophilic for long periods of time. A thin PPAA film of about 5 nm was deposited on the stamps, which increased the hydrophilicity, and which remained stable for at least several months. These surface-modified stamps were used to transfer polar inks by microCP. The employed microCP schemes are as follows: (a) a second generation of dendritic ink having eight dialkyl sulfide end groups to fabricate patterns on gold substrates by positive microCP, (b) fluorescent guest molecules on beta-cyclodextrin (beta-CD) printboards on glass employing host-guest recognition, and (c) Lucifer Yellow ethylenediamine resulting in covalent patterning on an aldehyde-terminated glass surface. All experiments resulted in an excellent performance of all three PPAA-coated stamp materials to transfer the polar inks from the stamp surface to gold and glass substrates by microCP, even from aqueous solutions.     

High-precision microcontact printing of interchangeable stamps using an integrated kinematic coupling

Microcontact printing (μCP) is a rapid, inexpensive way to create microscale chemical or biochemical patterns on a target surface. This microstamping method can be used to selectively modify a wide array of surface properties, from wettability and protein adsorption to chemical etch susceptibility. However, controlling the absolute location of features created with microcontact printing is difficult; this lack of precision makes it challenging to integrate with other microfabrication methods or to create complex, multi-chemical patterns on a single surface. In this research, we demonstrate a novel method of controlling the placement of microcontact printing stamps by using an integrated kinematic coupling device. This technique relies on mechanical reference points for rapid, optics-free registry of the stamp and allows μCP stamps to be quickly removed and replaced or even exchanged with submicron repeatability.     

High-speed microcontact printing

We have demonstrated microcontact printing (muCP) of self-assembled monolayers in the millisecond regime. The contact formation and separation of the stamp and substrate was studied with high-speed video recordings. Using high ink concentrations and contact times as short as 1 ms, we printed monolayers of hexadecanethiol on Au, which served as a selective etch resist. High-speed muCP yields defect-free monolayers that are independent of the dimensions of the printed patterns, have high contrast between printed and unprinted areas, and enable perfect reproducibility of prints.     

Diffusion of alkanethiols in PDMS and its implications on microcontact printing (muCP)

n-Alkanethiols HS-(CH2)n-CH3 such as hexadecanethiol (HDT, n = 15), octadecanethiol (ODT, n = 17), and eicosanethiol (ECT, n = 19) have been shown to provide highly protective etch resists on microcontact-printed noble metals. As the quality of the printed pattern strongly depends on the mobility of the ink compound, we focused on understanding the diffusion behavior of HDT, ODT, and ECT in poly(dimethylsiloxane) (PDMS) stamps. We used a commercial PDMS material (Sylgard184), which is commonly used for microcontact printing (muCP), and a custom-synthesized one with a higher modulus. On the basis of linear-diffusion experiments, which maintained realistic printing conditions, we showed that the ink transport in the stamp follows Fick's law of diffusion. We then determined the diffusion coefficient by analytical and numerical modeling of the diffusion experiments. Numerical calculations were carried out with the finite-difference method applying more realistic boundary conditions (ink adsorption). Values for the diffusion coefficients of the three ink compounds in the two different PDMS materials all are on the order of (4-7) x 10(-7) cm2 s(-1). The scope and limits of the mathematical models are discussed. To demonstrate the potential of such models for microcontact printing, we simulate multiple printing cycles of an inked stamp and compare the results with experimental data.     

A method of printing uniform protein lines by using flat PDMS stamps

In this paper, we report a method of printing uniform protein lines on glass slides by using UV-treated flat PDMS stamps. Unlike traditional microcontact printing (μCP) which requires microstructured PDMS stamps, this μCP method only requires a flat PDMS stamp, an UV lamp and a number of straight needles. Our results show that lines of bovine serum albumin (BSA), immunoglobin (IgG), anti-biotin, anti-human IgG and anti-mouse IgG can be printed evenly on glass slides by using this μCP method. We also demonstrate that the printed protein lines are suitable for applications such as microfluidic immunoassays.     

Biocatalytic microcontact printing

Immobilized biocatalytic lithography is presented as an application of soft lithography. In traditional microcontact printing, diffusion limits resolution of pattern transfer. By using an immobilized catalyst, the lateral resolution of microcontact printing would depend only on the length and flexibility of the tether (<2 nm) as opposed to diffusion (>100 nm). In the work, exonuclease reversibly immobilized on a relief-patterned stamp is used to ablate ssDNA monolayers Percent of ablation was determined via confocal fluorescence microscopy to be approximately 70%.     

Printing chemical gradients

We describe a method to exploit the mass-transfer limitations of microcontact printing for the fabrication of surfaces with well-defined, arbitrarily shaped composition variations. An analysis of the transport processes reveals that the printing of hexadecanethiol (HDT) from poly(dimethylsiloxane) is purely diffusion-controlled. Stamps with geometries that enhance surface-normal diffusion paths therefore allow not only the contours, but also the local density of self-assembled monolayers to be controlled. We use stamps with variable thickness and uniform ink concentration to print HDT density gradients on gold, depleting the stamps during the process. In the second step, a perfluorinated thiol fills the vacancies in the partial monolayer to form a two-component gradient that we analyze by means of X-ray photoelectron spectroscopy and spectroscopic ellipsometry. Linear and radial gradients are shown here as examples for a wide range of geometries that can be fabricated with high precision using the method.     

Binary self-assembled monolayers of alkanethiols on gold: deposition from solution versus microcontact printing and the study of surface nanobubbles

The coadsorption of alkanethiols on noble metals has been recognized for a long time as a suitable means of affording surfaces with systematically varied wettability and other properties. In this article, we report on a comparative study of the composition of the mixed self-assembled monolayers (SAMs) obtained (i) by the coadsorption of octadecanethiol (ODT) and 16-mercaptohexadecanoic acid (MHDA) from ethanol and chloroform onto gold substrates and (ii) by microcontact printing using poly(dimethyl siloxane) (PDMS) stamps. SAMs prepared by coadsorption from solution showed a preferential adsorption of ODT for both solvents, but this trend was reversed in microcontact-printed SAMs when using chloroform as a solvent, as evidenced by contact angle and Fourier transform infrared (FTIR) spectroscopy measurements. An approximately linear relationship between the static contact angle and the degree of swelling with different solvents was observed, which suggests that the surface composition can be controlled by the interaction of the solvent and the PDMS elastomer. The altered preference is attributed to the different partitioning of the two thiols into solvent-swelled PDMS, as shown by (1)H NMR spectroscopy. Finally, molecularly mixed binary SAMs on ODT and MHDA on template-stripped gold were applied to study the effect of surface nanobubbles on wettability by atomic force microscopy (AFM). With a decreasing macroscopic contact angle measured through water, the nanoscopic contact angle was found to decrease as well.