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.
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.
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.
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.
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.
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