A microfluidic, dual-purpose sensor for in vitro detection of Enterobacteriaceae and biotinylated antibodies.

In this paper, we present a versatile, dual-purpose sensor for in vitro detection of Enterobacteriaceae (e.g. Escherichia coli) and biotinylated antibodies (e.g. IgG rabbit polyclonal antibodies), based on different detection principles for each bioanalyte. These bioanalytes are tagged individually with functionalized magnetic microparticles, suspended into a static fluid and injected into a microfluidic channel. Without the need for bulk or complicated pumping systems, the functionalized microparticles are set in motion by a magnetic force exerted on them by integrated microconductors. The fundamental detection principle is the decrease in the velocity of the microparticles that are loaded with the respective bioanalyte, due to factors inhibiting their motion. The velocity of the unloaded, bare microparticles is used as a reference. We discovered a novel mechanism on which the constrained particle motion is based; in the case of E. coli, the inhibiting factor is the enhanced Stokes' drag force due to the greater volume and altered hydrodynamic shape, whereas in the case of biotinylated antibodies, it is the increased friction force at the interface between the modified microparticle and the biosensor's surface. Friction force is for the first time employed in a scheme for resolving biomolecules. Integrated magnetic microsensors are used for the velocity measurements by detecting the microparticles' stray field. Moreover, we developed a biocompatible, easy to implement and reliable surface modification that practically diminishes the problem of bioadhesion on the sensor's surface.

A single-molecule approach to explore binding, uptake and transport of cancer cell targeting nanotubes.

In the past decade carbon nanotubes (CNTs) have been widely studied as a potential drug-delivery system, especially with functionality for cellular targeting. Yet, little is known about the actual process of docking to cell receptors and transport dynamics after internalization. Here we performed single-particle studies of folic acid (FA) mediated CNT binding to human carcinoma cells and their transport inside the cytosol. In particular, we employed molecular recognition force spectroscopy, an atomic force microscopy based method, to visualize and quantify docking of FA functionalized CNTs to FA binding receptors in terms of binding probability and binding force. We then traced individual fluorescently labeled, FA functionalized CNTs after specific uptake, and created a dynamic 'roadmap' that clearly showed trajectories of directed diffusion and areas of nanotube confinement in the cytosol. Our results demonstrate the potential of a single-molecule approach for investigation of drug-delivery vehicles and their targeting capacity.

Combined single cell AFM manipulation and TIRFM for probing the molecular stability of multilayer fibrinogen matrices.

Adsorption of fibrinogen on various surfaces produces a nanoscale multilayer matrix, which strongly reduces the adhesion of platelets and leukocytes with implications for hemostasis and blood compatibility of biomaterials. The nonadhesive properties of fibrinogen matrices are based on their extensibility, ensuing the inability to transduce strong mechanical forces via cellular integrins and resulting in weak intracellular signaling. In addition, reduced cell adhesion may arise from the weaker associations between fibrinogen molecules in the superficial layers of the matrix. Such reduced stability would allow integrins to pull fibrinogen molecules out of the matrix with comparable or smaller forces than required to break integrin-fibrinogen bonds. To examine this possibility, we developed a method based on the combination of total internal reflection fluorescence microscopy, single cell manipulation with an atomic force microscope and microcontact printing to study the transfer of fibrinogen molecules out of a matrix onto cells. We calculated the average fluorescence intensities per pixel for wild-type HEK 293 (HEK WT) and HEK 293 cells expressing leukocyte integrin Mac-1 (HEK Mac-1) before and after contact with multilayered matrices of fluorescently labeled fibrinogen. For contact times of 500 s, HEK Mac-1 cells show a median increase of 57% of the fluorescence intensity compared to 6% for HEK WT cells. The results suggest that the integrin Mac-1-fibrinogen interactions are stronger than the intermolecular fibrinogen interactions in the superficial layer of the matrix. The low mechanical stability of the multilayer fibrinogen surface may contribute to the reduced cell adhesive properties of fibrinogen-coated substrates. We anticipate that the described method can be applied to various cell types to examine their integrin-mediated adhesion to the extracellular matrices with a variable protein composition.

Mapping the intracellular distribution of carbon nanotubes after targeted delivery to carcinoma cells using confocal Raman imaging as a label-free technique.

The uptake of carbon nanotubes (CNTs) by mammalian cells and their distribution within cells is being widely studied in recent years due to their increasing use for biomedical purposes. The two main imaging techniques used are confocal fluorescence microscopy and transmission electron microscopy (TEM). The former, however, requires labeling of the CNTs with fluorescent dyes, while the latter is a work-intensive technique that is unsuitable for in situ bio-imaging. Raman spectroscopy, on the other hand, presents a direct, straightforward and label-free alternative. Confocal Raman microscopy can be used to image the CNTs inside cells, exploiting the strong Raman signal connected to different vibrational modes of the nanotubes. In addition, cellular components, such as the endoplasmic reticulum and the nucleus, can be mapped. We first validate our method by showing that only when using the CNTs' G band for intracellular mapping accurate results can be obtained, as mapping of the radial breathing mode (RBM) only shows a small fraction of CNTs. We then take a closer look at the exact localization of the nanotubes inside cells after folate receptor-mediated endocytosis and show that, after 8-10 h incubation, the majority of CNTs are localized around the nucleus. In summary, Raman imaging has enormous potential for imaging CNTs inside cells, which is yet to be fully realized.