A DNA-based platform was developed to address fundamental aspects of early stages of cell signaling in living cells. By site-directed sorting of differently encoded, protein-decorated DNA origami structures on DNA microarrays, we combine the advantages of the bottom-up self-assembly of protein-DNA nanostructures and top-down micropatterning of solid surfaces to create multiscale origami structures as interface for cells (MOSAIC). In a proof-of-principle, we use this technology to analyze the activation of epidermal growth factor (EGF) receptors in living MCF7 cells using DNA origami structures decorated on their surface with distinctive nanoscale arrangements of EGF ligand entities. MOSAIC holds the potential to present to adhered cells well-defined arrangements of ligands with full control over their number, stoichiometry, and precise nanoscale orientation. It therefore promises novel applications in the life sciences, which cannot be tackled by conventional technologies.

Acute subcellular protein targeting is a powerful tool to study biological networks. However, signaling at the plasma membrane is highly dynamic, making it difficult to study in space and time. In particular, sustained local control of molecular function is challenging owing to the lateral diffusion of plasma membrane targeted molecules. Herein we present "molecular activity painting" (MAP), a novel technology which combines photoactivatable chemically induced dimerization (pCID) with immobilized artificial receptors. The immobilization of artificial receptors by surface-immobilized antibodies blocks lateral diffusion, enabling rapid and stable "painting" of signaling molecules and their activity at the plasma membrane with micrometer precision. Using this method, we show that painting of the RhoA-myosin activator GEF-H1 induces patterned acto-myosin contraction inside living cells.

Glioblastoma is the most common primary tumor of the brain and has few long-term survivors. The local and systemic immunosuppressive environment created by glioblastoma allows it to evade immunosurveillance. Myeloid-derived suppressor cells (MDSCs) are a critical component of this immunosuppression. Understanding mechanisms of MDSC formation and function are key to developing effective immunotherapies. In this study, we developed a novel model to reliably generate human MDSCs from healthy-donor CD14+ monocytes by culture in human glioma-conditioned media. Monocytic MDSC frequency was assessed by flow cytometry and confocal microscopy. The resulting MDSCs robustly inhibited T cell proliferation. A cytokine array identified multiple components of the GCM potentially contributing to MDSC generation, including Monocyte Chemoattractive Protein-1, interleukin-6, interleukin-8, and Macrophage Migration Inhibitory Factor (MIF). Of these, Macrophage Migration Inhibitory Factor is a particularly attractive therapeutic target as sulforaphane, a naturally occurring MIF inhibitor derived from broccoli sprouts, has excellent oral bioavailability. Sulforaphane inhibits the transformation of normal monocytes to MDSCs by glioma-conditioned media in vitro at pharmacologically relevant concentrations that are non-toxic to normal leukocytes. This is associated with a corresponding increase in mature dendritic cells. Interestingly, sulforaphane treatment had similar pro-inflammatory effects on normal monocytes in fresh media but specifically increased immature dendritic cells. Thus, we have used a simple in vitro model system to identify a novel contributor to glioblastoma immunosuppression for which a natural inhibitor exists that increases mature dendritic cell development at the expense of myeloid-derived suppressor cells when normal monocytes are exposed to glioma conditioned media.

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.

Multi-color patterning by polymer pen lithography (PPL) was used to fabricate covalently immobilized fluorophore and oligonucleotide arrays with up to five different components. The oligonucleotide arrays offer a virtually unlimited inventory of orthogonal binding tags for self-assembly of proteins as demonstrated by use of the arrays to monitor cell-protein interactions of MCF7 cells.

Gradient patterns comprising bioactive compounds over comparably (in regard to a cell size) large areas are key for many applications in the biomedical sector, in particular, for cell screening assays, guidance, and migration experiments. Polymer pen lithography (PPL) as an inherent highly parallel and large area technique has a great potential to serve in the fabrication of such patterns. We present strategies for the printing of functional phospholipid patterns via PPL that provide tunable feature size and feature density gradients over surface areas of several square millimeters. By controlling the printing parameters, two transfer modes can be achieved. Each of these modes leads to different feature morphologies. By increasing the force applied to the elastomeric pens, which increases the tip-surface contact area and boosts the ink delivery rate, a switch between a dip-pen nanolithography (DPN) and a microcontact printing (μCP) transfer mode can be induced. A careful inking procedure ensuring a homogeneous and not-too-high ink-load on the PPL stamp ensures a membrane-spreading dominated transfer mode, which, used in combination with smooth and hydrophilic substrates, generates features with constant height, independently of the applied force of the pens. Ultimately, this allows us to obtain a gradient of feature sizes over a mm substrate, all having the same height on the order of that of a biological cellular membrane. These strategies allow the construction of membrane structures by direct transfer of the lipid mixture to the substrate, without requiring previous substrate functionalization, in contrast to other molecular inks, where structure is directly determined by the printing process itself. The patterns are demonstrated to be viable for subsequent protein binding, therefore adding to a flexible feature library when gradients of protein presentation are desired.

The patterned immobilization of chemosensors into nano/microarrays has often boosted utilization in diagnostics and environmental sensing applications. While this is a standard approach for biosensors, e.g., with antibodies, other proteins, and DNA, arraying is not yet adopted widely for supramolecular chemosensors which are still predominantly used in solution systems. Here we introduce the patterned immobilization of cucurbit[n]urils (CBn) into multiplexed microarrays and elucidate their prospects for the advancement of surface-bound indicator-displacement assays to detect small molecule analytes. The microarrays were generated by microchannel cantilever spotting of functionalized CBn and subsequent self-assembly of the corresponding indicator dyes from solution. Enhanced sensitivity of surface-bound microarrays was established in demonstrations with small bioactive metabolites (spermine, amantadine, and cadaverine) compared to bulk assays. Furthermore, the integration of the CBn/indicator microarrays into microfluidic channels provides an efficient route for real-time monitoring of the sensing process, allows easier handling, and reduces need for analyte volume. The concept was further extended to differential sensing of analytes on diplex or multiplex CBn/indicator microarrays, opening up a route for multicomponent sensing of small molecule analytes in complex liquids.

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.