We present a handheld biosensor system for the label-free and specific multiplexed detection of several biomarkers employing a spectrometer-free imaging measurement system. A photonic crystal surface functionalized with multiple specific ligands forms the optical transducer. The photonic crystal slab is fabricated on a glass substrate by replicating a periodic grating master stamp with a period of 370 nm into a photoresist via nanoimprint lithography and deposition of a 70-nm titanium dioxide layer. Capture molecules are coupled covalently and drop-wise to the photonic crystal surface. With a simple camera and imaging optics the surface-normal transmission is detected. In the transmission spectrum guided-mode resonances are observed that shift due to protein binding. This shift is observed as an intensity change in the green color channel of the camera. Non-functionalized image sections are used for continuous elimination of background drift. In a first experiment we demonstrate the specific and time-resolved detection of 90.0 nm CD40 ligand antibody, 90.0 nM EGF antibody, and 500 nM streptavidin in parallel on one sensor chip. In a second experiment, aptamers with two different spacer lengths are used as receptor. The binding kinetics with association and dissociation of 250 nM thrombin and regeneration of the sensor surface with acidic tris-HCl-buffer (pH 5.0) is presented for two measurement cycles.

Porous hydrogel scaffolds are ideal candidates for mimicking cellular microenvironments, regarding both structural and mechanical aspects. We present a novel strategy to use uniquely designed ceramic networks as templates for generating hydrogels with a network of interconnected pores in the form of microchannels. The advantages of this new approach are the high and guaranteed interconnectivity of the microchannels, as well as the possibility to produce channels with diameters smaller than 7 μm. Neither of these assets can be ensured with other established techniques. Experiments using the polyacrylamide substrates produced with our approach have shown that the migration of human pathogenic Acanthamoeba castellanii trophozoites is manipulated by the microchannel structure in the hydrogels. The parasites can even be captured inside the microchannel network and removed from their incubation medium by the porous polyacrylamide, indicating the huge potential of our new technique for medical, pharmaceutical, and tissue engineering applications.

Summary Background: Mechanosensing of cells, particularly the cellular response to substrates with different elastic properties, has been discovered in recent years, but almost exclusively in mammalian cells. Much less attention has been paid to mechanosensing in other cell systems, such as in eukaryotic human pathogens. Results: We report here on the influence of substrate stiffness on the adhesion of the human pathogen Acanthamoebae castellanii (A. castellanii). By comparing the cell adhesion area of A. castellanii trophozoites on polydimethylsiloxane (PDMS) substrates with different Young’s moduli (4 kPa, 29 kPa, and 128 kPa), we find significant differences in cell adhesion area as a function of substrate stiffness. In particular, the cell adhesion area of A. castellanii increases with a decreasing Young’s modulus of the substrate. Conclusion: The dependence of A. castellanii adhesion on the elastic properties of the substrate is the first study suggesting a mechanosensory effect for a eukaryotic human pathogen. Interestingly, the main targets of A. castellanii infections in the human body are the eye and the brain, i.e., very soft environments. Thus, our study provides first hints towards the relevance of mechanical aspects for the pathogenicity of eukaryotic parasites.

We designed and evaluated a microfluidic test chip for human blood filtration and imaging label-free detection of multiple biomarkers. The microfluidic chip has a total size of 75 mm x 25 mm x 2 mm. It is realized as an assembly of a plastic chip and a functionalized photonic crystal slab on a glass substrate. The plastic chip contains capillary channels, a filter membrane and a cavity open on one side. The photonic crystal chip is bonded with an adhesive foil to the open cavity. Human blood filtration is demonstrated. We determined that fluorescently labelled particles of diameter 3µm or larger are filtered out by the system. Refractometric measurements are performed with the test chip in combination with a compact imaging read-out system to investigate the system response to refractive index changes. Flow dynamics in the sensor cavity are imaged for replacing water by isopropanol. Finally, the binding of 500 nM biotin dissolved in phosphate buffer saline to a photonic crystal surface locally functionalized with streptavidin is demonstrated.

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