RESUMEN

We introduce a principle of parallel optical processing to an optofluidic lab-on-a-chip. During electrophoretic separation, the ultra-low limit of detection achieved with our set-up allows us to record fluorescence from covalently end-labeled DNA molecules. Different sets of exclusively color-labeled DNA fragments-otherwise rendered indistinguishable by spatio-temporal coincidence-are traced back to their origin by modulation-frequency-encoded multi-wavelength laser excitation, fluorescence detection with a single ultrasensitive, albeit color-blind photomultiplier, and Fourier analysis decoding. As a proof of principle, fragments obtained by multiplex ligation-dependent probe amplification from independent human genomic segments, associated with genetic predispositions to breast cancer and anemia, are simultaneously analyzed.

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RESUMEN

By applying integrated-waveguide laser excitation to an optofluidic chip, fluorescently labeled DNA molecules of 12 or 17 different sizes are separated by CE with high operating speed and low sample consumption of approximately 600 pL. When detecting the fluorescence signals of migrating DNA molecules with a PMT, the LOD is as low as 2.1 pM. In the diagnostically relevant size range (approximately 150-1000 base-pairs) the molecules are separated with reproducibly high sizing accuracy (> 99%) and the plug broadening follows Poissonian statistics. Variation of the power dependence of migration time on base-pair size--probably with temperature and condition of the sieving gel matrix--indicates that the capillary migration cannot be described by a simple physical law. Integrated-waveguide excitation of a 12-microm narrow microfluidic segment provides a spatio-temporal resolution that would, in principle, allow for a 20-fold better accuracy than the currently supported by state-of-the-art electrophoretic separation in microchips, thereby demonstrating the potential of this integrated optical approach to fulfill the resolution demands of future electrophoretic microchips.

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We present a simple approach in electrophoretic DNA separation and fluorescent monitoring that allows to identify the insertion or deletion of base-pairs in DNA probe molecules from genetic samples, and to perform intrinsic calibration/referencing for highly accurate DNA analysis. The principle is based on dual-point, dual-wavelength laser-induced fluorescence excitation using one or two excitation windows at the intersection of integrated waveguides and microfluidic channels in an optofluidic chip and a single, color-blind photodetector, resulting in a limit of detection of ~200 pM for single-end-labeled DNA molecules. The approach using a single excitation window is demonstrated experimentally, while the option exploiting two excitation windows is proposed theoretically.

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Using femtosecond laser writing, optical waveguides were monolithically integrated into a commercial microfluidic lab-on-a-chip device, with the waveguides intersecting a microfluidic channel. Continuous-wave laser excitation through these optical waveguides confines the excitation window to a width of 12 microm, enabling high-resolution monitoring of the passage of different types of fluorescent analytes when migrating and being separated in the microfluidic channel by microchip capillary electrophoresis. Furthermore, we demonstrate on-chip-integrated waveguide excitation and detection of a biologically relevant species, fluorescently labeled DNA molecules, during microchip capillary electrophoresis. Well-controlled plug formation as required for on-chip integrated capillary electrophoresis separation of DNA molecules, and the combination of waveguide excitation and a low limit of detection, will enable monitoring of extremely small quantities with high spatial resolution.

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Galectin-1 is the homodimeric form of a protein, which is present in a dynamic equilibrium with the beta-galactoside monomeric form and has potent anti-inflammatory and immunomodulating effects. These favorable effects are probably related to the induction of apoptosis in activated T cells and the induction of IL-10, which have been demonstrated to be characteristic for the dimeric form of the protein. Based on these findings it can be speculated that the in vivo effects of galectin-1 can be improved by the generation of stable galectin-1 homodimers (dGal). To test this hypothesis we produced leucine-zipper based stable galectin-1 homodimers and tested its apoptosis inducing effects on MOLT-4 cells and its immunomodulatory effects in vitro on PBMC of five independent donors. Phosphatidylserine exposure and a drop in mitochondrial membrane potential was strongly enhanced on MOLT-4 cells upon treatment with dGal as compared to wtGal. The minimal effective concentration was 20-fold reduced as compared to the minimal effective wtGal concentration. dGal showed enhanced induction of IL-10 on total PBMC as compared to treatment with wild-type protein (wtGal). The minimal effective dGal concentration was 100-fold lower than that of wtGal. Of the purified cell populations monocytes are the strongest IL-10 producers, whereas T cells induce IL-10 at a lower level and no induction is observed in B cells. Besides induction of IL-10, dGal caused an increase in IL-1beta production in all donors and a reduction of IL-2 production in 3 out of 5 donors, whereas no consistent changes were observed for other inflammatory cytokines. In summary, we demonstrated that dGal shows enhanced effects at strongly reduced concentrations. Application of dGal may therefore serve as an improved treatment of chronic inflammatory diseases.

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Galectin-1, a beta-galactoside binding protein that can occur as both a monomer and a homodimer, binds to leucocyte membrane antigens such as CD7, CD43, and CD45, and has immune-regulatory functions in several animal models of autoimmune disease. However, its mechanism of action is only partially understood. In this study, a marked increase in IL-10 mRNA and protein levels was demonstrated in non-activated and activated CD4(+) and CD8(+) T-cells, following treatment with a high concentration (dimeric form), but not a low concentration (monomeric form), of recombinant galectin-1 protein. IL-10 is known to suppress TH1 type immune responses and upregulation of IL-10 may thus contribute to the immune-regulatory function of galectin-1. Galectin-1 was strongly expressed on the endothelial cells of human kidney allografts, suggesting a role in the regulation of immune responses in transplantation. Administration of high concentrations of galectin-1 may be a useful tool in the treatment of T-cell-mediated diseases.

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