Falko Brinkmann

Lipid multilayer gratings

The interaction of electromagnetic waves with matter can be controlled by structuring the matter on the scale of the wavelength of light, and various photonic components have been made by structuring materials using top-down or bottom-up approaches. Dip-pen nanolithography is a scanning-probe-based fabrication technique that can be used to deposit materials on surfaces with high resolution and, when carried out in parallel, with high throughput. Here, we show that lyotropic optical diffraction gratings--composed of biofunctional lipid multilayers with controllable heights between approximately 5 and 100 nm--can be fabricated by lipid dip-pen nanolithography. Multiple materials can be simultaneously written into arbitrary patterns on pre-structured surfaces to generate complex structures and devices, allowing nanostructures to be interfaced by combinations of top-down and bottom-up fabrication methods. We also show that fluid and biocompatible lipid multilayer gratings allow label-free and specific detection of lipid-protein interactions in solution. This biosensing capability takes advantage of the adhesion properties of the phospholipid superstructures and the changes in the size and shape of the grating elements that take place in response to analyte binding.

Interdigitated multicolored bioink micropatterns by multiplexed polymer pen lithography.

Multiplexing, i.e., the application and integration of more than one ink in an interdigitated microscale pattern, is still a challenge for microcontact printing (μCP) and similar techniques. On the other hand there is a strong demand for interdigitated patterns of more than one protein on subcellular to cellular length scales in the lower micrometer range in biological experiments. Here, a new integrative approach is presented for the fabrication of bioactive microarrays and complex multi-ink patterns by polymer pen lithography (PPL). By taking advantage of the strength of microcontact printing (μCP) combined with the spatial control and capability of precise repetition of PPL in an innovative way, a new inking and writing strategy is introduced for PPL that enables true multiplexing within each repetitive subpattern. Furthermore, a specific ink/substrate platform is demonstrated that can be used to immobilize functional proteins and other bioactive compounds over a biotin-streptavidin approach. This patterning strategy aims specifically at application by cell biologists and biochemists addressing a wide range of relevant pattern sizes, easy pattern generation and adjustment, the use of only biofriendly, nontoxic chemicals, and mild processing conditions during the patterning steps. The retained bioactivity of the fabricated cm(2) area filling multiprotein patterns is demonstrated by showing the interaction of fibroblasts and neurons with multiplexed structures of fibronectin and laminin or laminin and ephrin, respectively.

Large-Scale Parallel Surface Functionalization of Goblet-type Whispering Gallery Mode Microcavity Arrays for Biosensing Applications

A novel surface functionalization technique is presented for large-scale selective molecule deposition onto whispering gallery mode microgoblet cavities. The parallel technique allows damage-free individual functionalization of the cavities, arranged on-chip in densely packaged arrays. As the stamp pad a glass slide is utilized, bearing phospholipids with different functional head groups. Coated microcavities are characterized and demonstrated as biosensors.

Toxic and non-toxic aggregates from the SBMA and normal forms of androgen receptor have distinct oligomeric structures

Hormone-dependent aggregation of the androgen receptor (AR) with a polyglutamine (polyQ) stretch amplification (>38) is considered to be the causative agent of the neurodegenerative disorder spinal and bulbar muscular atrophy (SBMA), consistent with related neurodegenerative diseases involving polyQ-extended proteins. In spite of the widespread acceptance of this common causal hypothesis, little attention has been paid to its apparent incompatibility with the observation of AR aggregation in healthy individuals with no polyQ stretch amplification. Here we used atomic force microscopy (AFM) to characterize sub-micrometer scale aggregates of the wild-type (22 glutamines) and the SBMA form (65 glutamines), as well as a polyQ deletion mutant (1 glutamine) and a variant with a normal length polyQ stretch but with a serine to alanine double mutation elsewhere in the protein. We used a baculovirus-insect cell expression system to produce full-length proteins for these structural analyses. We related the AFM findings to cytotoxicity as measured by expression of the receptors in Drosophila motoneurons or in neuronal cells in culture. We found that the pathogenic AR mutants formed oligomeric fibrils up to 300-600nm in length. These were clearly different from annular oligomers 120-180nm in diameter formed by the nonpathogenic receptors. We could also show that melatonin, which is known to ameliorate the pathological phenotype in the fly model, caused polyQ-extended AR to form annular oligomers. Further comparative investigation of these reproducibly distinct toxic and non-toxic oligomers could advance our understanding of the molecular basis of the polyQ pathologies.

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.

A Versatile Microarray Platform for Capturing Rare Cells.

Analyses of rare events occurring at extremely low frequencies in body fluids are still challenging. We established a versatile microarray-based platform able to capture single target cells from large background populations. As use case we chose the challenging application of detecting circulating tumor cells (CTCs) – about one cell in a billion normal blood cells. After incubation with an antibody cocktail, targeted cells are extracted on a microarray in a microfluidic chip. The accessibility of our platform allows for subsequent recovery of targets for further analysis. The microarray facilitates exclusion of false positive capture events by co-localization allowing for detection without fluorescent labelling. Analyzing blood samples from cancer patients with our platform reached and partly outreached gold standard performance, demonstrating feasibility for clinical application. Clinical researchers free choice of antibody cocktail without need for altered chip manufacturing or incubation protocol, allows virtual arbitrary targeting of capture species and therefore wide spread applications in biomedical sciences.

Densely Packed Microgoblet Laser Pairs for Cross-Referenced Biomolecular Detection

Microgoblet laser pairs are presented for cross‐referenced on‐chip biomolecular sensing. Parallel readout of the microlasers facilitates effective mutual filtering of highly localized refractive index and temperature fluctuations in the analyte. Cross‐referenced detection of two different types of proteins and complete chemical transducer reconfiguration is demonstrated. Selective surface functionalization of the individual lasers with high spatial accuracy is achieved by aligned microcontact stamping.

Ultra-large scale AFM of lipid droplet arrays: investigating the ink transfer volume in dip pen nanolithography.

There are only few quantitative studies commenting on the writing process in dip-pen nanolithography with lipids. Lipids are important carrier ink molecules for the delivery of bio-functional patters in bio-nanotechnology. In order to better understand and control the writing process, more information on the transfer of lipid material from the tip to the substrate is needed. The dependence of the transferred ink volume on the dwell time of the tip on the substrate was investigated by topography measurements with an atomic force microscope (AFM) that is characterized by an ultra-large scan range of 800 × 800 μm(2). For this purpose arrays of dots of the phospholipid1,2-dioleoyl-sn-glycero-3-phosphocholine were written onto planar glass substrates and the resulting pattern was imaged by large scan area AFM. Two writing regimes were identified, characterized of either a steady decline or a constant ink volume transfer per dot feature. For the steady state ink transfer, a linear relationship between the dwell time and the dot volume was determined, which is characterized by a flow rate of about 16 femtoliters per second. A dependence of the ink transport from the length of pauses before and in between writing the structures was observed and should be taken into account during pattern design when aiming at best writing homogeneity. The ultra-large scan range of the utilized AFM allowed for a simultaneous study of the entire preparation area of almost 1 mm(2), yielding good statistic results.

Patterning of quantum dots by dip-pen and polymer pen nanolithography

Abstract We present a direct way of patterning CdSe/ ZnS quantum dots by dip-pen nanolithography and polymer pen lithography. Mixtures of cholesterol and phospholipid 1,2-dioleoyl-sn-glycero-3 phosphocholine serve as biocompatible carrier inks to facilitate the transfer of quantum dots from the tips to the surface during lithography. While dip-pen nanolithography of quantum dots can be used to achieve higher resolution and smaller pattern features (approximately 1 μm), polymer pen lithography is able to address intermediate pattern scales in the low micrometre range. This allows us to combine the advantages of micro contact printing in large area and massive parallel patterning, with the added flexibility in pattern design inherent in the DPN technique.

Highly integrated biophotonics towards all-organic lab-on-chip systems

For sensing with lab-on-chip systems, the use of highly integrated photonic components is a key ingredient for high sensitivity and short response time. Here, we present the efficient integration of organic semiconductor distributed feedback lasers based on Alq3:DCM and deep ultraviolet induced waveguides into a Poly(methyl methacrylate) (PMMA) substrate. The optimized coupling of laser light from the organic semiconductor lasers into the waveguides is discussed. On-chip coupling of laser light at 645 nm to waveguides is demonstrated. Utilizing mass production technologies and simple processes with only a few different materials paves the way for low-cost all-organic chips. In particular, a specific sensing concept is introduced: deep ultraviolet induced waveguides and nanostructured phospholipid gratings, which are bottom-up assembled using Dip-Pen Nanolithography, are combined to form a grating coupler with a grating period of 700 nm. Light of different wavelength is decoupled under different angles by the grating coupler to demonstrate its functional capability. Biosensing with this device is discussed based on a model system.