Czech Contribution to LOFT

We describe the current status of the Czech contribution to the ESA LOFT space mission, with emphasis on technical aspects. Expertise available in the Czech Republic will play a positive role in the LOFT project and related developments

Detection of immunoglobulins in a laser induced fluorescence system utilizing polydimethysiloxane microchips with advanced surface and optical properties

We developed an automated laser induced fluorescence system utilizing microfluidic chips for detection and quantification of immunoglobulins. Microchips were fabricated from polydimethysiloxane (PDMS) using the so-called “prepolymerization technique.” The microchip structure helped minimize the effects of PDMS autofluorescence and light scattering. Furthermore, a thin and uniform PDMS layer forming the top of the microchip enabled proper focusing and collection of the excitation beam and the emitted fluorescence, respectively. The developed system was tested for the detection of mouse immunoglobulins. The capturing antibodies were immobilized on internal microchannel walls in the form of a polyelectrolyte. We clearly show that this immobilization technique, if correctly realized, gives results with high reproducibility. After sample incubation and washing, secondary antibodies labeled by fluorescein isothiocyanate were introduced into microchannels to build a detectable complex. We show that mouse antibodies can be quantified in a wide concentration range, 0.01–100 μg ml−1. The lower detection limit was below 0.001 μg ml−1 (6.7 pM). The developed laser induced fluorescence (LIF) apparatus is relatively cheap and easy to construct. The total cost of the developed LIF detector is lower than a typical price of plate readers. If compared to classical ELISA (enzyme linked immunosorbent assay) plate systems, the detection of immunoglobulins or other proteins in the developed PDMS microfluidic device brings other important benefits such as reduced time demands (10 min incubation) and low reagent consumption (less than 1 μl). The cost of the developed PDMS chips is comparable with the price of commercial ELISA plates. The main troubleshooting related to the apparatus development is also discussed in order to help potential constructors

Microfluidic chip for fast bioassays—evaluation of binding parameters

A seven channel polystyrene (PS) microchip has been constructed using a micromilling machine and a high-temperature assembling. Protein A (PA) has been immobilized by a passive sorption on the microchannel walls. Two bioaffinity assays with human immunoglobulin G (hIgG) as a ligand have been carried out. (i) PA as the receptor and fluorescently labeled hIgG (FITC-hIgG) as the ligand, (ii) PA as the receptor with hIgG as the quantified ligand and fluorescently labeled goat anti-human IgG (FITC-gIgG) as the secondary ligand. One incubation step of the assays took only 5 min instead of hours typical for enzyme-linked immunosorbent assay applications. Calibration curves of the dependence of a fluorescence signal on the hIgG concentration in a sample have been obtained in one step due to a parallel arrangement of microchannels. A mathematical model of the PA-FITC-hIgG complex formation in the chip has been developed. The values of the kinetic constant of the PA-FITC-hIgG binding (kon=5.5 m3 mol−1 s−1) and the equilibrium dissociation constant of the formed complex (Kd≤3×10−6 mol m−3) have been obtained by fitting to experimental data. The proposed microchip enables fast evaluation of kinetic and equilibrium constants of ligand-receptor bioaffinity pairs and the ligand quantification. As the use of microfluidic chips for immunoassays is often limited by price, we used procedures and chemicals that allow for an inexpensive construction and operation of the microdevice, e.g., temperature assembling as a fabrication technique, detection via an ordinary digital camera, nonspecific polystyrene as a substrate, passive sorption of biomolecules as an immobilization technique, etc