Surface-Patterned DNA Origami Rulers Reveal Nanoscale Distance Dependency of the Epidermal Growth Factor Receptor Activation.

The nanoscale arrangement of ligands can have a major effect on the activation of membrane receptor proteins and thus cellular communication mechanisms. Here we report on the technological development and use of tailored DNA origami-based molecular rulers to fabricate "Multiscale Origami Structures As Interface for Cells" (MOSAIC), to enable the systematic investigation of the effect of the nanoscale spacing of epidermal growth factor (EGF) ligands on the activation of the EGF receptor (EGFR). MOSAIC-based analyses revealed that EGF distances of about 30-40 nm led to the highest response in EGFR activation of adherent MCF7 and Hela cells. Our study emphasizes the significance of DNA-based platforms for the detailed investigation of the molecular mechanisms of cellular signaling cascades.

Patterned immobilization of polyoxometalate-loaded mesoporous silica particles via amine-ene Michael additions on alkene functionalized surfaces.

Polyoxometalates (POM) are anionic oxoclusters of early transition metals that are of great interest for a variety of applications, including the development of sensors and catalysts. A crucial step in the use of POM in functional materials is the production of composites that can be further processed into complex materials, e.g. by printing on different substrates. In this work, we present an immobilization approach for POMs that involves two key processes: first, the stable encapsulation of POMs in the pores of mesoporous silica nanoparticles (MSPs) and, second, the formation of microstructured arrays with these POM-loaded nanoparticles. Specifically, we have developed a strategy that leads to water-stable, POM-loaded mesoporous silica that can be covalently linked to alkene-bearing surfaces by amine-Michael addition and patterned into microarrays by scanning probe lithography (SPL). The immobilization strategy presented facilitates the printing of hybrid POM-loaded nanomaterials onto different surfaces and provides a versatile method for the fabrication of POM-based composites. Importantly, POM-loaded MSPs are useful in applications such as microfluidic systems and sensors that require frequent washing. Overall, this method is a promising way to produce surface-printed POM arrays that can be used for a wide range of applications.

A supramolecular cucurbit[8]uril-based rotaxane chemosensor for the optical tryptophan detection in human serum and urine.

Sensing small biomolecules in biofluids remains challenging for many optical chemosensors based on supramolecular host-guest interactions due to adverse interplays with salts, proteins, and other biofluid components. Instead of following the established strategy of developing alternative synthetic binders with improved affinities and selectivity, we report a molecular engineering approach that addresses this biofluid challenge. Here we introduce a cucurbit[8]uril-based rotaxane chemosensor feasible for sensing the health-relevant biomarker tryptophan at physiologically relevant concentrations, even in protein- and lipid-containing human blood serum and urine. Moreover, this chemosensor enables emission-based high-throughput screening in a microwell plate format and can be used for label-free enzymatic reaction monitoring and chirality sensing. Printed sensor chips with surface-immobilized rotaxane-microarrays are used for fluorescence microscopy imaging of tryptophan. Our system overcomes the limitations of current supramolecular host-guest chemosensors and will foster future applications of supramolecular sensors for molecular diagnostics.

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.

Correlated Study of Material Interaction Between Capillary Printed Eutectic Gallium Alloys and Gold Electrodes.

Liquid metals (LMs) play a growing role in flexible electronics and connected applications. Here, LMs come into direct contact with metal electrodes thus allowing for corrosion and additional alloying, potentially compromising device stability. Nevertheless, comprehensive studies on the interfacial interaction of the materials are still sparse. Therefore, a correlated material interaction study of capillary-printed Galinstan (eutetic alloy of Ga/In/Sn) with gold surfaces and electrodes is conducted. Comprehensive application of optical microscopy, vertical scanning interferometry, scanning electron microscopy/spectroscopy, x-ray photon spectroscopy, and atomic force microscopy allow for an in depth characterization of the spreading process of LM lines on gold films, revealing the differential spread of the different LM components and the formation of intermetallic nanostructures on the surface of the surrounding gold film. A model for the growth process based on the penetration of LM along the gold film grain boundaries is proposed based on the obtained time-dependent characterization. The distribution of gold, Galinstan, and intermetallic phases in a gold wire dipped into LM is observed using X-ray nano tomography as a complementary view on the internal nanostructure. Finally, resistance measurements on LM lines connecting gold electrodes over time allow to estimate the influence on the material interaction on electronic applications.

FluidFM-Based Fabrication of Nanopatterns: Promising Surfaces for Platelet Storage Application.

Platelets are cell fragments from megakaryocytes devoid of the cell nucleus. They are highly sensitive and easily activated by nonphysiological surfaces. Activated platelets have an intrinsic mechanism to release various proteins that participate in multiple pathways, initiating the platelet activation cascade. Surface-induced platelet activation is a challenge encountered during platelet storage, which eventually leads to aggregation of platelets and can thereby result in the degradation of the platelet concentrates. We have previously reported that surface-induced platelet activation can be minimized by either modifying their contact surfaces with polymers or introducing nanogroove patterns underneath the platelets. Here, we investigated the response of platelets to various nanotopographical surfaces printed using fluidic force microscopy (FluidFM). We found that the hemispherical array (grid) and hexagonal tile (hive) structures caused a reduction of surface stiffness, which leads to an inhibition of platelet adhesion. Our results reveal that nanopatterns enable the inhibition of platelet activation on surfaces, thus implying that development in nanotexturing of storage bags can extend the lifetime of platelet concentrates.

Multiplexed Covalent Patterns on Double-Reactive Porous Coating.

We have conceptualized and demonstrated an approach based on the combination of hydrophobicity, a substrate-independent dip coating as porous material with double residual chemical reactivities for implementing multiplexed, miniaturized and unclonable bulk-infused patterns of different fluorophores following distinct reaction pathways. The embedded hydrophobicity (∼102°) restricted the unwanted spreading of beaded aqueous ink on the coating. The constructions of micropatterns on porous dip-coating via ink-jet printing or microchannel cantilever spotting offered orthogonal read-out and remained readable even after removal of the exterior of the coating.

Integration of Biofunctional Molecules into 3D-Printed Polymeric Micro-/Nanostructures.

Three-dimensional printing at the micro-/nanoscale represents a new challenge in research and development to achieve direct printing down to nanometre-sized objects. Here, FluidFM, a combination of microfluidics with atomic force microscopy, offers attractive options to fabricate hierarchical polymer structures at different scales. However, little is known about the effect of the substrate on the printed structures and the integration of (bio)functional groups into the polymer inks. In this study, we printed micro-/nanostructures on surfaces with different wetting properties, and integrated molecules with different functional groups (rhodamine as a fluorescent label and biotin as a binding tag for proteins) into the base polymer ink. The substrate wetting properties strongly affected the printing results, in that the lateral feature sizes increased with increasing substrate hydrophilicity. Overall, ink modification only caused minor changes in the stiffness of the printed structures. This shows the generality of the approach, as significant changes in the mechanical properties on chemical functionalization could be confounders in bioapplications. The retained functionality of the obtained structures after UV curing was demonstrated by selective binding of streptavidin to the printed structures. The ability to incorporate binding tags to achieve specific interactions between relevant proteins and the fabricated micro-/nanostructures, without compromising the mechanical properties, paves a way for numerous bio and sensing applications. Additional flexibility is obtained by tuning the substrate properties for feature size control, and the option to obtain functionalized printed structures without post-processing procedures will contribute to the development of 3D printing for biological applications, using FluidFM and similar dispensing techniques.

Direct-Write Patterning of Biomimetic Lipid Membranes In Situ with FluidFM.

The creation of biologically inspired artificial membranes on substrates with custom size and in close proximity to each other not only provides a platform to study biological processes in a simplified manner, but they also constitute building blocks for chemical or biological sensors integrated in microfluidic devices. Scanning probe lithography tools such as dip-pen nanolithography (DPN) have opened a new paradigm in this regard, although they possess some inherent drawbacks like the need to operate in air environment or the limited choice of lipids that can be patterned. In this work, we propose the use of the fluid force microscopy (FluidFM) technology to fabricate biomimetic membranes without losing the multiplexing capability of DPN but gaining flexibility in lipid inks and patterning environment. We shed light on the driving mechanisms of the FluidFM-mediated lithography processes in air and liquid. The obtained results should prompt the creation of more realistic biomimetic membranes with arbitrary complex phospholipid mixtures, cholesterol, and potential functional membrane proteins directly patterned in physiological environment.

A multiplexed phospholipid membrane platform for curvature sensitive protein screening.

The curvature of lipid membranes plays a key role in many relevant biological processes such as membrane trafficking, vesicular budding and host-virus interactions. In vitro studies on the membrane curvature of simplified biomimetic models in the nanometer range are challenging, due to their complicated nanofabrication processes. In this work, we propose a simple and low-cost platform for curvature sensitive protein screening, prepared through scanning probe lithography (SPL) methods, where lipid bilayer patches of different compositions can be multiplexed onto substrate areas with tailored local curvature. The curvature is imposed by anchoring nanoparticles of the desired size to the substrate prior to lithography. As a proof of principle, we demonstrate that a positive curvature membrane sensitive protein derived from the BAR domain of Nadrin2 binds selectively to lipid patches patterned on substrate areas coated with 100 nm nanoparticles. The platform opens up a path for screening curvature-dependent protein-membrane interaction studies by providing a flexible and easy to prepare substrate with control over lipid composition and membrane curvature.

Rapid Capture of Cancer Extracellular Vesicles by Lipid Patch Microarrays.

Extracellular vesicles (EVs) contain various bioactive molecules such as DNA, RNA, and proteins, and play a key role in the regulation of cancer progression. Furthermore, cancer-associated EVs carry specific biomarkers and can be used in liquid biopsy for cancer detection. However, it is still technically challenging and time consuming to detect or isolate cancer-associated EVs from complex biofluids (e.g., blood). Here, a novel EV-capture strategy based on dip-pen nanolithography generated microarrays of supported lipid membranes is presented. These arrays carry specific antibodies recognizing EV- and cancer-specific surface biomarkers, enabling highly selective and efficient capture. Importantly, it is shown that the nucleic acid cargo of captured EVs is retained on the lipid array, providing the potential for downstream analysis. Finally, the feasibility of EV capture from patient sera is demonstrated. The demonstrated platform offers rapid capture, high specificity, and sensitivity, with only a small need in analyte volume and without additional purification steps. The platform is applied in context of cancer-associated EVs, but it can easily be adapted to other diagnostic EV targets by use of corresponding antibodies.

High-precision tabletop microplotter for flexible on-demand material deposition in printed electronics and device functionalization.

Microstructuring, in particular, the additive functionalization of surfaces with, e.g., conductive or bioactive materials plays a crucial role in many applications in sensing or printed electronics. Mostly, the lithography steps are made prior to assembling functionalized surfaces into the desired places of use within a bigger device as a microfluidic channel or an electronic casing. However, when this is not possible, most lithography techniques struggle with access to recessed or inclined/vertical surfaces for geometrical reasons. In particular, for "on-the-fly" printing aiming to add microstructures to already existing devices on demand and maybe even for one-time trials, e.g., in prototyping, a flexible "micropencil" allowing for direct write under direct manual control and on arbitrarily positioned surfaces would be highly desirable. Here, we present a highly flexible, micromanipulator-based setup for capillary printing of conductive and biomaterial ink formulations that can address a wide range of geometries as exemplified on vertical, recessed surfaces and stacked 3D scaffolds as models for hard to access surfaces. A wide range of feature sizes from tens to hundreds of micrometer can be obtained by the choice of capillary sizes and the on-demand in situ writing capabilities are demonstrated with completion of a circuit structure by gold line interconnects deposited with the setup.

Enhanced Stability of Lipid Structures by Dip-Pen Nanolithography on Block-Type MPC Copolymer.

Biomimetic lipid membranes on solid supports have been used in a plethora of applications, including as biosensors, in research on membrane proteins or as interfaces in cell experiments. For many of these applications, structured lipid membranes, e.g., in the form of arrays with features of different functionality, are highly desired. The stability of these features on a given substrate during storage and in incubation steps is key, while at the same time the substrate ideally should also exhibit antifouling properties. Here, we describe the highly beneficial properties of a 2-methacryloyloxyethyl phosphorylcholine (MPC) copolymer for the stability of supported lipid membrane structures generated by dip-pen nanolithography with phospholipids (L-DPN). The MPC copolymer substrates allow for more stable and higher membrane stack structures in comparison to other hydrophilic substrates, like glass or silicon oxide surfaces. The structures remain highly stable under immersion in liquid and subsequent incubation and washing steps. This allows multiplexed functionalization of lipid arrays with antibodies via microchannel cantilever spotting (µCS), without the need of orthogonal binding tags for each antibody type. The combined properties of the MPC copolymer substrate demonstrate a great potential for lipid-based biomedical sensing and diagnostic platforms.

Evaluation of Microfluidic Ceiling Designs for the Capture of Circulating Tumor Cells on a Microarray Platform.

The capture of circulating tumor cells (CTCs) is still a challenging application for microfluidic chips, as these cells are rare and hidden in a huge background of blood cells. Here, different microfluidic ceiling designs in regard to their capture efficiency for CTCs in model experiments and more realistic conditions of blood samples spiked with a clinically relevant amount of tumor cells are evaluated. An optimized design for the capture platform that allows highly efficient recovery of CTCs from size-based pre-enriched samples under realistic conditions is obtained. Furthermore, the viability of captured tumor cells as well as single cell recovery for downstream genomic analysis is demonstrated. Additionally, the authors' findings underline the importance of evaluating rational design rules for microfluidic devices based on theoretical models by application-specific experiments.

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.

How Does Chemistry Influence Liquid Wettability on Liquid-Infused Porous Surface?

Design of Nepenthes pitcher-inspired slippery liquid-infused porous surface (SLIPS) appeared as an important avenue for various potential and practically relevant applications. In general, hydrophobic base layers were infused with selected liquid lubricants for developing chemically inert SLIPS. Here, in this current study, an inherently hydrophilic (soaked beaded water droplet with ∼20° within a couple of minutes), porous and thick (above 200 µm) polymeric coating, loaded with readily chemically reactive acrylate moieties yielded a chemically reactive SLIPS, where residual acrylate groups in the synthesized hydrophilic and porous interface rendered stability to the infused lubricants. The chemically reactive SLIPS is capable of reacting with the solution of primary amine-containing nucleophiles in organic solvent through 1,4-conjugate addition reaction, both in the presence (referred as "in situ" modification) and absence (denoted as pre-modification) of lubricated phase in the porous polymeric coating. Such amine reactive SLIPS was further extended to (1) examining the impact of different chemical modifications on the performance of SLIPS and (2) developing a spatially selective and "in situ" postmodification with primary amine-containing nucleophiles through 1,4-conjugate addition reaction. Moreover, the chemically reactive SLIPS was capable of sustaining various physical abrasions and prolonged (minimum 10 days) exposure to complex and harsh aqueous phases, where infused lubricants protect the residual acrylate groups from harsh aqueous exposures. Such, principle will be certainly useful for spatially selective covalent immobilization of water-insoluble functional molecules/polymers directly from organic solvents, which would be of potential interest for various applied and fundamental contexts.

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.

Aptamer Conformation-Cooperated Enzyme-Assisted Surface-Enhanced Raman Scattering Enabling Ultrasensitive Detection of Cell Surface Protein Biomarkers in Blood Samples.

Developing novel strategies for sensitive and specific detection of protein biomarkers is a field of active research. Here, we report an ultrasensitive biosensor to detect protein tyrosine kinase-7 (PTK7), an important protein biomarker on the cell surface, by aptamer conformation-cooperated enzyme-assisted surface-enhanced Raman scattering (SERS) (ACCESS) technology. Our approach features a synergistic combination of the conformational alteration of the anglerfish aptamer triggered by the recognition of the membrane protein (PTK7) and Exo III enzyme-assisted nucleic acid amplification. It transduces the specific binding events between the aptamer and PTK7 protein into dramatically improved SERS signals. Sensitive and specific detection of PTK7 protein has been demonstrated both in the solution and directly on the surface of live CCRF-CEM cells, with a limit of detection better than the commercial enzyme-linked immunosorbent assay method by nearly 5 orders of magnitude. As a flexible, ultrasensitive, and specific approach, ACCESS promises important applications in clinical diagnostics, where only a very limited amount of the biological sample is available.