Image-based fluidic sorting system for automated Zebrafish egg sorting into multiwell plates.

The global demand for the reduction of animal testing has led to the emergence of Zebrafish eggs/larvae as model organisms to replace current adult animal testing in, for example, toxicity testing. Because of the egg size (diameter 1.6mm) and the relatively easy maintenance of Zebrafish farms the eggs also offer high-throughput screening (HTS). However, the current bottleneck for HTS is the cost-efficient placing of individual organisms into single wells of a multiwell plate (MWP). The system presented here is capable of storing, sorting, and placing individual organisms in a highly reproducible manner. In about 11 min a complete 96-MWP is filled, which corresponds to about 8 sec per egg. The survival rate of fertilized transgenic and wild-type eggs was comparable to the one of the control (control 6.7%, system 7.6%). Furthermore, it was also possible to place dechorionated eggs into individual wells. The results demonstrate that the cost efficient system works gentle and reliable enough to disburden scientists from the exhausting and monotonous job of placing single eggs into single wells, such that they can concentrate on the scientific aspects of their experiments and create results with a higher statistical relevance.

Fully automated microinjection system for Xenopus laevis oocytes with integrated sorting and collection.

Microinjection is the most flexible transfection method in terms of choice of reagents to inject into cells. But this method lacks the high throughput to compete with less flexible methods like chemical- or viral-based approaches. Various approaches have been pursued to increase the throughput by automating the microinjection process. However, these approaches focused solely on the microinjection itself and disregarded the tasks before and after the injection, which also belong to the critical time path of the whole process, that is, sorting out viable cells from a cell suspension, placing the cell for injection, and collecting the cell after the injection. In the approach with our XenoFactor, we demonstrate a system capable of running the whole process automatically. By optimizing the XenoFactor for Xenopus laevis oocytes, we could demonstrate the successful automated injection. Starting from a suspension with a mixture of defolliculated oocytes at different stages and quality levels, the manual approach requires 1 day in total for the preparation of 400 microinjected oocytes. The XenoFactor takes only 4h for the same amount and delivers injected oocytes of reproducible quality and without the fatigue symptoms experienced during the manual approach.

Lab-on-a-chip for multiplexed biosensing of residual antibiotics in milk.

A multiplexed immunoassay-based antibiotic sensing device integrated in a lab-on-a-chip format is described. The approach is multidisciplinary and involves the convergent development of a multi-antibiotic competitive immunoassay based on sensitive wavelength interrogated optical sensor (WIOS) technology and a polymer-based self-contained microfluidic cartridge. Immunoassay solutions are pressure-driven through external and concerted actuation of a single syringe pump and multiposition valve. Moreover, the use of a novel photosensitive material in a 'one step' fabrication process allowed the rapid fabrication of microfluidic components and interconnection port simultaneously. Pre-filled microfluidic cartridges were used as binary response rapid tests for the simultaneous detection of three antibiotic families - sulfonamides, fluoroquinolones and tetracyclines - in raw milk. For test interpretation, any signal lower than the threshold value obtained for the corresponding Maximum Residue Limit (MRL) concentration (100 microg L(-1)) was considered negative for a given antibiotic. The reliability of the multiplexed detection system was assessed by way of a validation test carried out on a series of six blind milk samples. A test accuracy of 95% was calculated from this experiment. The whole immunoassay procedure is fast (less than 10 minutes) and easy to handle (automated actuation).

Templated growth of carbon nanotubes with controlled diameters using organic-organometallic block copolymers with tailored block lengths.

Block copolymer thin films fabricated from polystyrene-polyferrocenylsilane (PS-b-PFS) block copolymers on silicon substrates were used as precursors of well-ordered, nanosized growth catalysts for carbon nanotubes (CNTs). The size of the catalytic domains was tuned by changing the molecular weight of the block copolymer, enabling control of the diameter of the CNTs grown from these substrates. CNT growth on catalytic substrates with larger organometallic domain sizes, using acetylene as a carbon source, resulted in enhanced amounts of CNT deposition compared to smaller PFS domains, which exhibited low catalytic activity. The inner and outer diameters of the multi-walled CNTs obtained were typically 8 and 16 nm, respectively, and were not influenced by the catalytic domain sizes. Various annealing strategies in inert or in hydrogen atmosphere were investigated. The use acetylene with an additional hydrogen flow as gas feed resulted in a significant increase in deposition on all PS-b-PFS decorated substrates. Under these conditions, the CNT diameters could be controlled by the catalyst domain sizes, resulting in decreasing diameters with decreasing domain sizes. Multiwalled CNTs with inner and outer diameters of 4 and 7 nm, respectively, and a narrow diameter distribution were obtained.

Fast immobilization of probe beads by dielectrophoresis-controlled adhesion in a versatile microfluidic platform for affinity assay.

The use of probe beads for lab-on-chip affinity assays is very interesting from a practical point of view. It is easier to handle and trap beads than molecules in microfluidic systems. We present a method for the immobilization of probe beads at defined areas on a chip using dielectrophoresis (DEP)-controlled adhesion. The method is fast, i.e., it takes between 10 and 120 s--depending on the protocol--to functionalize a chip surface at defined areas. The method is versatile, i.e., it works for beads with different types of probe molecule coatings. The immobilization is irreversible, i.e., the retained beads are able to withstand high flow velocities in a flow-through device even after the DEP voltage is turned off, thus allowing the use of conventional high-conductivity analyte buffers in the following assay procedure. We demonstrate the on-chip immobilization of fluorescent beads coated with biotin, protein A, and goat-antimouse immunoglobulin G (IgG). The number of immobilized beads at an electrode array can be determined from their fluorescence signal. Further, we use this method to demonstrate the detection of streptavidin and mouse IgG. Finally, we demonstrate the feasibility of the parallel detection of different analyte molecules on the same chip.

Development of preparation methods for and insights obtained from atomic force microscopy of fluid spaces in cortical bone.

Several preparation methods were developed to investigate the dimensions and surface structure of fluid spaces within cortical bone, using atomic force microscopy (AFM). Of special interest was the morphology of the lacunocanalicular system, which serves as a conduit between osteocytes encased in bone tissue, the intramedullary cavity, blood vessels running through the bone, and the periosteal surface of bone. Fracture and the removal of either the mineral or the organic component is a method by which each component can be investigated at a very high resolution in situ. Although fractured bone was too rough to image details of the lacunocanalicular system, post-treatment with ethylene diamine tetraacetic acid (EDTA) or papain allowed for investigation of the collagen matrix or the mineral crystals of bone, respectively. Cut and polished bone was smooth enough for identifying the lacunae of bone using AFM, but unambiguous differentiation between the canaliculi and cracks in the bone surface was not possible. However, when the lacunocanalicular system was filled with polymethylmethacrylate (PMMA), it was possible to image casts of the lacunocanalicular system by selectively etching away the surrounding bone matrix. Using this method, we identified individual canaliculi and measured their dimensions. Furthermore, by carefully etching away the bone matrix in successive etches, it was shown that the wall structure of the canaliculus is dominated by collagen fibrils. These observations have important implications for fluid flow in bone.