3D Nanolithography by Means of Lipid Ink Spreading Inhibition.

While patterning 2D metallic nanostructures are well established through different techniques, 3D printing still constitutes a major bottleneck on the way to device miniaturization. In this work a fluid phase phospholipid ink is used as a building block for structuring with dip-pen nanolithography. Following a bioinspired approach that relies on ink-spreading inhibition, two processes are presented to build 2D and 3D metallic structures. Serum albumin, a widely used protein with an innate capability to bind to lipids, is the key in both processes. Covering the sample surface with it prior to lipid writing, anchors lipids on the substrate, which ultimately allows the creation of highly stable 3D lipid-based scaffolds to build metallic structures.

Synergies between Surface Microstructuring and Molecular Nanopatterning for Controlling Cell Populations on Polymeric Biointerfaces.

Polymeric biointerfaces are already being used extensively in a wide set of biomedical devices and systems. The possibility of controlling cell populations on biointerfaces may be essential for connecting biological systems to synthetic materials and for researching relevant interactions between life and matter. In this study, we present and analyze synergies between an innovative approach for surface microstructuring and a molecular nanopatterning procedure of recent development. The combined set of techniques used may be instrumental for the development of a new generation of functional polymeric biointerfaces. Eukaryotic cell cultures placed upon the biointerfaces developed, both before and after molecular patterning, help to validate the proposal and to discuss the synergies between the surface microstructuring and molecular nanopatterning techniques described in the study. Their potential role in the production of versatile polymeric biointerfaces for lab- and organ-on-a-chip biodevices and towards more complex and biomimetic co-culture systems and cell cultivation set-ups are also examined.

Thioacetate-Based Initiators for the Synthesis of Thiol-End-Functionalized Poly(2-oxazoline)s.

New functional initiators for the cationic ring-opening polymerization of 2-alkyl-2-oxazolines are described to introduce a thiol moiety at the α terminus. Both tosylate and nosylate initiators carrying a thioacetate group are obtained in multigram scale, from commercial reagents in two steps, including a phototriggered thiol-ene radical addition. The nosylate derivative gives access to a satisfying control over the cationic ring-opening polymerization of 2-ethyl-2-oxazoline, with dispersity values lower than 1.1 during the entire course of the polymerization, until full conversion. Cleavage of the thioacetate end group is rapidly achieved using triazabicyclodecene, thereby leading to a mercapto terminus. The latter gives access to a new subgeneration of α-functional poly(2-oxazoline)s (butyl ester, N-hydroxysuccinimidyl ester, furan) by Michael addition with commercial (meth)acrylates. The amenability of the mercapto-poly(2-ethyl-2-oxazoline) for covalent surface patterning onto acrylated surfaces is demonstrated in a microchannel cantilever spotting (µCS) experiment, characterized by X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary-ion mass spectrometry (ToF-SIMS).

Covalently Modulated and Transiently Visible Writing: Rational Association of Two Extremes of Water Wettabilities.

Anticounterfeiting measures are of ever-increasing importance in society, e.g., for securing the authenticity of and the proof of origin for medical drugs. Here, an arms race of counterfeiters and valid manufacturers is taking place, resulting in the need of hard-to-forget, yet easy-to-read out marks. Anticounterfeiting measures based on micropatterns-while being attractive for their need in not widely available printing methods while still being easily read out with fairly common basic optical equipment-are often limited by being too easy to be destroyed by wear or handling. Here, nature-inspired wettability is rationally exploited for developing an unprecedented anticounterfeiting method, where hidden information can be only identified under direct exposures to an aqueous phase or mist and disappears again on air-drying the interface. A chemically reactive and hierarchically featured dip coating, capable of spatially selective covalent modification with primary amine containing small molecules, is developed for abrasion-tolerant patterning interfaces with two extremes of water wettabilities, i.e., superhydrophilicity and superhydrophobicity. Arbitrary handwriting with glucamine followed by chemical modification with octadecylamine, provided "invisible" text on the synthesized interface. The glucamine-treated region selectively becomes optically transparent and superhydrophilic due to rapid infiltration of the aqueous phase on exposure to liquid water or mist. The remaining interface remains opaque and superhydrophobic due to metastable entrapment of air. The hidden text became transiently and reversibly visible by the naked eye under exposure to liquid water/mist. Furthermore, microchannel-cantilever spotting (µCS) is adopted for demonstrating well-defined chemical patterning on the microscale. These patterns are at the same time highly resistant against wear and scratching because of the bulk functionalization, retaining the wetting properties (and thus pattern readout) even on serious abrasion. Such a simple synthesis of spatially controlled, direct, and covalently modulated wettability could be useful for various applied and fundamental contexts.

Evaluation of click chemistry microarrays for immunosensing of alpha-fetoprotein (AFP).

The level of cancer biomarkers in cells, tissues or body fluids can be used for the prediction of the presence of cancer or can even indicate the stage of the disease. Alpha-fetoprotein (AFP) is the most commonly used biomarker for early screening and diagnosis of hepatocellular carcinoma (HCC). Here, a combination of three techniques (click chemistry, the biotin-streptavidin-biotin sandwich strategy and the use of antigen-antibody interactions) were combined to implement a sensitive fluorescent immunosensor for AFP detection. Three types of functionalized glasses (dibenzocyclooctyne- (DBCO-), thiol- and epoxy-terminated surfaces) were biotinylated by employing the respective adequate click chemistry counterparts (biotin-thiol or biotin-azide for the first class, biotin-maleimide or biotin-DBCO for the second class and biotin-amine or biotin-thiol for the third class). The anti-AFP antibody was immobilized on the surfaces via a biotin-streptavidin-biotin sandwich technique. To evaluate the sensing performance of the differently prepared surfaces, fluorescently labeled AFP was spotted onto them via microchannel cantilever spotting (µCS). Based on the fluorescence measurements, the optimal microarray design was found and its sensitivity was determined.

Site-Specific Surface Functionalization via Microchannel Cantilever Spotting (µCS): Comparison between Azide-Alkyne and Thiol-Alkyne Click Chemistry Reactions.

Different types of click chemistry reactions are proposed and used for the functionalization of surfaces and materials, and covalent attachment of organic molecules. In the present work, two different catalyst-free click approaches, namely azide-alkyne and thiol-alkyne click chemistry are studied and compared for the immobilization of microarrays of azide or thiol inks on functionalized glass surfaces. For this purpose, the surface of glass is first functionalized with dibenzocyclooctyne-acid (DBCO-acid), a cyclooctyne with a carboxyl group. Then, the DBCO-terminated surfaces are functionalized via microchannel cantilever spotting with different fluorescent and nonfluorescent azide and thiol inks. Although both routes work reliably for surface functionalization, the protein binding experiments reveal that using a thiol-alkyne route will obtain the highest surface density of molecular immobilization in such spotting approaches. The obtained achievements and results from this work can be used for design and manufacturing of microscale patterns suitable for biomedical and biological applications.

Phospholipid arrays on porous polymer coatings generated by micro-contact spotting.

Nanoporous poly(2-hydroxyethyl methacrylate-co-ethylene dimethacrylate) (HEMA-EDMA) is used as a 3D mesh for spotting lipid arrays. Its porous structure is an ideal matrix for lipid ink to infiltrate, resulting in higher fluorescent signal intensity as compared to similar arrays on strictly 2D substrates like glass. The embedded lipid arrays show high stability against washing steps, while still being accessible for protein and antibody binding. To characterize binding to polymer-embedded lipids we have applied Streptavidin as well as biologically important biotinylated androgen receptor binding onto 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(cap biotinyl) (Biotinyl Cap PE) and anti-DNP IgE recognition of 2,4-dinitrophenyl[1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[6-[(2,4-dinitrophenyl)amino]hexanoyl] (DNP)] antigen. This approach adds lipid arrays to the range of HEMA polymer applications and makes this solid substrate a very attractive platform for a variety of bio-applications.

Click-Chemistry Based Allergen Arrays Generated by Polymer Pen Lithography for Mast Cell Activation Studies.

The profiling of allergic responses is a powerful tool in biomedical research and in judging therapeutic outcome in patients suffering from allergy. Novel insights into the signaling cascades and easier readouts can be achieved by shifting activation studies of bulk immune cells to the single cell level on patterned surfaces. The functionality of dinitrophenol (DNP) as a hapten in the induction of allergic reactions has allowed the activation process of single mast cells seeded on patterned surfaces to be studied following treatment with allergen specific Immunoglobulin E antibodies. Here, a click-chemistry approach is applied in combination with polymer pen lithography (PPL) to pattern DNP-azide on alkyne-terminated surfaces to generate arrays of allergen. The large area functionalization offered by PPL allows an easy incorporation of such arrays into microfluidic chips. In such a setup, easy handling of cell suspension, incubation process, and read-out by fluorescence microscopy will allow immune cell activation screening to be easily adapted for diagnostics and biomedical research.

Dip-pen nanolithography-assisted protein crystallization.

We demonstrate the use of dip-pen nanolithography (DPN) to crystallize proteins on surface-localized functionalized lipid layer arrays. DOPC lipid layers, containing small amounts of biotin-DOPE lipid molecules, were printed on glass substrates and evaluated in vapor diffusion and batch crystallization screening setups, where streptavidin was used as a model protein for crystallization. Independently of the crystallization system used and the geometry of the lipid layers, nucleation of streptavidin crystals occurred specifically on the DPN-printed biotinylated structures. Protein crystallization on lipid array patches is also demonstrated in a microfluidic chip, which opens the way toward high-throughput screening to find suitable nucleation and crystal growth conditions. The results demonstrate the use of DPN in directing and inducing protein crystallization on specific surface locations.

Localization and dynamics of glucocorticoid receptor at the plasma membrane of activated mast cells.

In addition to their actions in the cell nucleus, glucocorticoids exhibit rapid non-nuclear responses that are mechanistically not well understood. To explain these effects, the localization of a glucocorticoid receptor (GR) expressed in mast cells as a GFP fusion was analyzed after activation of the cells on allergenic lipid arrays. These arrays were produced on glass slides by dip-pen nanolithography (DPN) and total internal reflection (TIRF) microscopy was used to visualize the GR. A rapid glucocorticoid-independent and -dependent recruitment of the GR-GFP to the plasma cell membrane was observed following contact of the cells with the allergenic array. In addition, the mobility of the GR at the membrane was monitored by fluorescence recovery after photobleaching (FRAP) and shown to follow binding kinetics demonstrating interactions of the receptor with membrane-bound factors. Furthermore the recruitment of the GR to the cell membrane was shown to result in a glucocorticoid-mediated increase in Erk phosphorylation. This is evidenced by findings that destruction of the membrane composition of the mast cells by cholesterol depletion impairs the membrane localization of the GR and subsequent glucocorticoid-mediated enhancement of Erk phosphorylation. These results demonstrate a membrane localization and function of the GR in mast cell signaling.

Convergence of dip-pen nanolithography and acoustic biosensors towards a rapid-analysis multi-sample microsystem.

The present work demonstrates for the first time patterning of a ready-to-use biosensor with several different biomolecules using Dip-Pen Nanolithography (DPN) for the development of a procedure towards more rapid and efficient multi-sample detection. The biosensor platform used is based on a Surface Acoustic Wave (SAW) device integrated with a parallel-channel microfluidic module, termed as "microfluidics-on-SAW" ("µF-on-SAW"), for reproducible multi-sample analysis. Lipids with different functionalized head groups were patterned at distinct, microfluidic-formed rectangular domains with sharp edges all located on the same sensor surface; pattern quality was verified using a fluorescent microscope. The functionality of the head groups, the efficiency of the patterning method, and the suitability of DPN for the surface modification of the acoustic device were subsequently examined through acoustic experiments. The µF-on-SAW configuration was used to detect specific binding between the pre-patterned functionalized lipids with their corresponding biomolecules. The achievement of an improved sensitivity (5-fold compared to previous acoustic configurations) and reduced preparation time by at least 2 h clearly indicates the suitability of DPN as a direct patterning method for ready-to-use acoustic sensor devices like the µF-on-SAW towards integrated, rapid-analysis, multi-sample biosensing microsystem development.

Allergen arrays for antibody screening and immune cell activation profiling generated by parallel lipid dip-pen nanolithography.

Multiple-allergen testing for high throughput and high sensitivity requires the development of miniaturized immunoassays that allow for a large test area and require only a small volume of the test analyte, which is often available only in limited amounts. Developing such miniaturized biochips containing arrays of test allergens needs application of a technique able to deposit molecules at high resolution and speed while preserving its functionality. Lipid dip-pen nanolithography (L-DPN) is an ideal technique to create such biologically active surfaces, and it has already been successfully applied for the direct, nanoscale deposition of functional proteins, as well as for the fabrication of biochemical templates for selective adsorption. The work presented here shows the application of L-DPN for the generation of arrays of the ligand 2,4-dinitrophenyl[1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[6-[(2,4-dinitrophenyl)amino]hexanoyl] (DNP)] onto glass surfaces as a model system for detection of allergen-specific Immunoglobin E (IgE) antibodies and for mast cell activation profiling.

Comparative height measurements of dip-pen nanolithography-produced lipid membrane stacks with atomic force, fluorescence, and surface-enhanced ellipsometric contrast microscopy.

Dip-pen nanolithography (DPN) with phospholipids has been shown to be a powerful tool for the generation of biologically active surface patterns, but screening of the obtained lithographic structures is still a bottleneck in the quality control of the prepared samples. Here we performed a comparative study with atomic force microscopy (AFM), fluorescence microscopy (FM), and surface-enhanced ellipsometric contrast (SEEC) microscopy of phospholipid membrane stacks consisting of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) with high admixing of 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[6-[(2,4-dinitrophenyl)amino]hexanoyl] (DNP Cap PE) produced by DPN. We present a structural model of membrane stacking based on the combined information gained from the three microscopic techniques. Domains of phase-separated DNP Cap PE can be detected at high DNP Cap PE admixing that are not present at medium or low admixings. While the optical methods allow for a high-throughput screening of lithographic structures (compared to AFM), it was found that, when relying on FM alone, artifacts due to phase-separation phenomena can be introduced in the case of thin membrane stacks.