Fast Inline Microscopic Computational Imaging.

Inline inspection is becoming an essential tool for industrial high-quality production. Unfortunately, the desired acquisition speeds and needs for high-precision imaging are often at the limit of what is physically possible, such as a large field of view at a high spatial resolution. In this paper, a novel light-field and photometry system is presented that addresses this trade off by combining microscopic imaging with special projection optics to generate a parallax effect. This inline microscopic system, together with an image processing pipeline, delivers high-resolution 3D images at high speeds, by using a lateral transport stage changing the optical perspective. Scanning speeds of up to 12 mm/s can be achieved at a depth resolution of 2.8 µm and a lateral sampling of 700 nm/pixel, suitable for inspection in high-quality manufacturing industry.

The trimer to monomer transition of Tumor Necrosis Factor-Alpha is a dynamic process that is significantly altered by therapeutic antibodies.

The cytokine tumor necrosis factor-alpha (TNF-α) readily forms homotrimers at sub-nM concentrations to promote inflammation. For the treatment of inflammatory diseases with upregulated levels of TNF-α, a number of therapeutic antibodies are currently used as scavengers to reduce the active TNF-α concentration in patients. Despite their clinical success, the mode-of-action of different antibody formats with regard to a stabilization of the trimeric state is not entirely understood. Here, we use a biosensor with dynamic nanolevers to analyze the monomeric and trimeric states of TNF-α together with the binding kinetics of therapeutic biologics. The intrinsic trimer-to-monomer decay rate k = 1.7 × 10-3 s-1 could be measured directly using a microfluidic system, and antibody binding affinities were analyzed in the pM range. Trimer stabilization effects are quantified for Adalimumab, Infliximab, Etanercept, Certolizumab, Golimumab for bivalent and monovalent binding formats. Clear differences in trimer stabilization are observed, which may provide a deeper insight into the mode-of-action of TNF-α scavengers.

Magnesium-Dependent Electrical Actuation and Stability of DNA Origami Rods.

Dynamic methods of biosensing based on electrical actuation of surface-tethered nanolevers require the use of levers whose movement in ionic liquids is well controllable and stable. In particular, mechanical integrity of the nanolevers in a wide range of ionic strengths will enable to meet the chemical conditions of a large variety of applications where the specific binding of biomolecular analytes is analyzed. Herein, we study the electrically induced switching behavior of different rodlike DNA origami nanolevers and compare to the actuation of simply double-stranded DNA nanolevers. Our measurements reveal a significantly stronger response of the DNA origami to switching of electrode potential, leading to a smaller potential change necessary to actuate the origami and subsequently to a long-term stable movement. Dynamic measurements in buffer solutions with different Mg2+ contents show that the levers do not disintegrate even at very low ion concentrations and constant switching stress and thus provide stable actuation performance. The latter will pave the way for many new applications without largely restricting application-specific environments.

Communication between N terminus and loop2 tunes Orai activation.

Ca2+ release-activated Ca2+ (CRAC) channels constitute the major Ca2+ entry pathway into the cell. They are fully reconstituted via intermembrane coupling of the Ca2+-selective Orai channel and the Ca2+-sensing protein STIM1. In addition to the Orai C terminus, the main coupling site for STIM1, the Orai N terminus is indispensable for Orai channel gating. Although the extended transmembrane Orai N-terminal region (Orai1 amino acids 73-91; Orai3 amino acids 48-65) is fully conserved in the Orai1 and Orai3 isoforms, Orai3 tolerates larger N-terminal truncations than Orai1 in retaining store-operated activation. In an attempt to uncover the reason for these isoform-specific structural requirements, we analyzed a series of Orai mutants and chimeras. We discovered that it was not the N termini, but the loop2 regions connecting TM2 and TM3 of Orai1 and Orai3 that featured distinct properties, which explained the different, isoform-specific behavior of Orai N-truncation mutants. Atomic force microscopy studies and MD simulations suggested that the remaining N-terminal portion in the non-functional Orai1 N-truncation mutants formed new, inhibitory interactions with the Orai1-loop2 regions, but not with Orai3-loop2. Such a loop2 swap restored activation of the N-truncation Orai1 mutants. To mimic interactions between the N terminus and loop2 in full-length Orai1 channels, we induced close proximity of the N terminus and loop2 via cysteine cross-linking, which actually caused significant inhibition of STIM1-mediated Orai currents. In aggregate, maintenance of Orai activation required not only the conserved N-terminal region but also permissive communication of the Orai N terminus and loop2 in an isoform-specific manner.

Regenerative biosensor for use with biotinylated bait molecules.

Label-free biosensors are ideally suited for the quantitative analysis of specific interactions among biomolecules or of biomolecules with drugs, as well as for quantitation of diagnostic markers in biofluids. In contrast to the label-dependent methods, a new assay for a particular prey molecule can be set up within few minutes by immobilizing the corresponding bait molecule on the sensor surface, using one of the common immobilization procedures. Unfortunately, the extensive application of label-free biosensors is still hampered by the fact that the immobilization of the bait molecule is usually irreversible; for that reason, a new chip (which is expensive) is required for every successful or futile attempt. Here, we present a general method for the switchable immobilization of biotinylated bait molecules on a new desthiobiotin surface, using wild-type streptavidin as a robust bridge between the chip and the biotinylated bait. The immobilization of the bait is very stable, so that many cycles of prey injection and subsequent prey removal can be carried out. For the latter, common reagents like HCl, Na2CO3, glycine buffer, or SDS are employed. When desired, however, streptavidin plus the biotinylated bait can be completely removed by 3min injections of biotin, guanidinium thiocyanate, pepsin, and SDS, which makes it possible to immobilize new biotinylated bait. The number of in situ regeneration cycles is unlimited during the lifetime of the chip (2-3 weeks). One chip can easily be shared by many users with unrelated tasks (as is typical in academics), or used for the fully automated screening of many different interactions (for example in pharmaceutical research). In comparison to other regenerative chips, the new chip surface has much wider applicability and all of its structural and functional parameters have been disclosed.

Detailed Evidence for an Unparalleled Interaction Mode between Calmodulin and Orai Proteins.

Calmodulin (CaM) binds most of its targets by wrapping around an amphipathic α-helix. The N-terminus of Orai proteins contains a conserved CaM-binding segment but the binding mechanism has been only partially characterized. Here, microscale thermophoresis (MST), surface plasmon resonance (SPR), and atomic force microscopy (AFM) were employed to study the binding equilibria, the kinetics, and the single-molecule interaction forces involved in the binding of CaM to the conserved helical segments of Orai1 and Orai3. The results consistently indicated stepwise binding of two separate target peptides to the two lobes of CaM. An unparalleled high affinity was found when two Orai peptides were dimerized or immobilized at high lateral density, thereby mimicking the close proximity of the N-termini in native Orai oligomers. The analogous experiments with smooth muscle myosin light chain kinase (smMLCK) showed only the expected 1:1 binding, confirming the validity of our methods.

Advanced portrayal of SMIL coating by allying CZE performance with in-capillary topographic and charge-related surface characterization.

A successive multiple ionic polymer layer (SMIL) coating composed of four layers improved the capillary electrophoretic separation of a recombinant major birch pollen allergen and closely related variants when poly(acrylamide-co-2-acrylamido-2-methyl-1-propansulfonate) (55% PAMAMPS) replaced dextran sulfate as terminal SMIL layer. 55% PAMAMPS decelerated the electroosmotic flow (EOF) due to its lower charge density. Atomic force microscopy (AFM) was used to investigate SMIL properties directly on the inner capillary surface and to relate them to EOF measurements and results of associated CZE separations of a mixture of model proteins and peptides that were performed in the same capillary. For the first time, AFM-based biosensing topography and recognition imaging mode (TREC) under liquid conditions was applied for a sequential characterization of the inner surface of a SMIL coated capillary after selected treatments including pristine SMIL, SMIL after contact with the model mixture, after alkaline rinsing, and the replenishment of the terminal polyelectrolyte layer. A cantilever with tip-tethered avidin was used to determine the charge homogeneity of the SMIL surface in the TREC mode. SMIL coated rectangular capillaries with 100 µm internal diameter assured accessibility of the inner surface for this cantilever type. Observed changes in CZE performance and EOF mobility during capillary treatment were also reflected by alterations in surface roughness and charge distribution of the SMIL coating. A renewal of the terminal SMIL layer restored the original surface properties of SMIL and the separation performance. The alliance of the novel TREC approach and CZE results allows for an improved understanding and a comprehensive insight in effects occurring on capillary coatings.

Curli mediate bacterial adhesion to fibronectin via tensile multiple bonds.

Many enteric bacteria including pathogenic Escherichia coli and Salmonella strains produce curli fibers that bind to host surfaces, leading to bacterial internalization into host cells. By using a nanomechanical force-sensing approach, we obtained real-time information about the distribution of molecular bonds involved in the adhesion of curliated bacteria to fibronectin. We found that curliated E. coli and fibronectin formed dense quantized and multiple specific bonds with high tensile strength, resulting in tight bacterial binding. Nanomechanical recognition measurements revealed that approximately 10 bonds were disrupted either sequentially or simultaneously under force load. Thus the curli formation of bacterial surfaces leads to multi-bond structural components of fibrous nature, which may explain the strong mechanical binding of curliated bacteria to host cells and unveil the functions of these proteins in bacterial internalization and invasion.

Mapping molecular adhesion sites inside SMIL coated capillaries using atomic force microscopy recognition imaging.

Capillary zone electrophoresis (CZE) is a powerful analytical technique for fast and efficient separation of different analytes ranging from small inorganic ions to large proteins. However electrophoretic resolution significantly depends on the coating of the inner capillary surface. High technical efforts like Successive Multiple Ionic Polymer Layer (SMIL) generation have been taken to develop stable coatings with switchable surface charges fulfilling the requirements needed for optimal separation. Although the performance can be easily proven in normalized test runs, characterization of the coating itself remains challenging. Atomic force microscopy (AFM) allows for topographical investigation of biological and analytical relevant surfaces with nanometer resolution and yields information about the surface roughness and homogeneity. Upgrading the scanning tip to a molecular biosensor by adhesive molecules (like partly inverted charged molecules) allows for performing topography and recognition imaging (TREC). As a result, simultaneously acquired sample topography and adhesion maps can be recorded. We optimized this technique for electrophoresis capillaries and investigated the charge distribution of differently composed and treated SMIL coatings. By using the positively charged protein avidin as a single molecule sensor, we compared these SMIL coatings with respect to negative charges, resulting in adhesion maps with nanometer resolution. The capability of TREC as a functional investigation technique at the nanoscale was successfully demonstrated.

Broadband 120 MHz Impedance Quartz Crystal Microbalance (QCM) with Calibrated Resistance and Quantitative Dissipation for Biosensing Measurements at Higher Harmonic Frequencies.

We developed an impedance quartz crystal microbalance (QCM) approach with the ability to simultaneously record mass changes and calibrated energy dissipation with high sensitivity using an impedance analyzer. This impedance QCM measures frequency shifts and resistance changes of sensing quartz crystals very stable, accurately, and calibrated, thus yielding quantitative information on mass changes and dissipation. Resistance changes below 0.3 Ω were measured with corresponding dissipation values of 0.01 µU (micro dissipation units). The broadband impedance capabilities allow measurements between 20 Hz and 120 MHz including higher harmonic modes of up to 11th order for a 10 MHz fundamental resonance frequency quartz crystal. We demonstrate the adsorbed mass, calibrated resistance, and quantitative dissipation measurements on two biological systems including the high affinity based avidin-biotin interaction and nano-assemblies of polyelectrolyte layers. The binding affinity of a protein-antibody interaction was determined. The impedance QCM is a versatile and simple method for accurate and calibrated resistance and dissipation measurements with broadband measurement capabilities for higher harmonics measurements.