Rodolphe Marie

DNA Catenation Maintains Structure of Human Metaphase Chromosomes

Mitotic chromosome structure is pivotal to cell division but difficult to observe in fine detail using conventional methods. DNA catenation has been implicated in both sister chromatid cohesion and chromosome condensation, but has never been observed directly. We have used a lab-on-a-chip microfluidic device and fluorescence microscopy, coupled with a simple image analysis pipeline, to digest chromosomal proteins and examine the structure of the remaining DNA, which maintains the canonical ‘X’ shape. By directly staining DNA, we observe that DNA catenation between sister chromatids (separated by fluid flow) is composed of distinct fibres of DNA concentrated at the centromeres. Disrupting the catenation of the chromosomes with Topoisomerase IIα significantly alters overall chromosome shape, suggesting that DNA catenation must be simultaneously maintained for correct chromosome condensation, and destroyed to complete sister chromatid disjunction. In addition to demonstrating the value of microfluidics as a tool for examining chromosome structure, these results lend support to certain models of DNA catenation organization and regulation: in particular, we conclude from our observation of centromere-concentrated catenation that spindle forces could play a driving role in decatenation and that Topoisomerase IIα is differentially regulated at the centromeres, perhaps in conjunction with cohesin.

Resonance Raman spectroscopy on whole blood in a microfluidic device with hydrodynamic cell-free layer creation (Conference Presentation)

We demonstrate resonance Raman spectroscopy in microfluidic channels for the analysis of whole blood. In particular, cell-free plasma layers are created in microfluidic whole blood flow by means of temporary hydrodynamic cell filter functionality. In-line confocal Raman spectroscopy is applied at the location of the created cell-free plasma layer and we detect free hemoglobin at diagnostic relevant hemolysis concentrations. Raman spectroscopy is a semi-quantitative method for chemical analysis. Due to the uniqueness of molecular vibrations, its selectivity is dependable. However, Raman scattering intensity is often too weak to be detected. This weakness can be overcome by resonance Raman spectroscopy where the laser excitation frequency is chosen corresponding to a dipole-allowed electronic transition of the molecule under study [1]. By employing resonance Raman spectroscopy we investigate bovine blood samples inside microfluidic channels in a micro-Raman setup, putting analytical emphasis on hemoglobin. Resonance Raman spectroscopy of hemoglobin was first demonstrated in 1972 [2] where aqueous solutions with concentrations of approximately 10-4 M were examined. Here we detect hemoglobin dissolved in bovine blood plasma inside 40μm deep PDMS channels. Most dominantly in our Raman spectra, the characteristic oscillatory mode of the central porphyrin ring structure of hemoglobin at 1375cm-1 appears. Despite the background in the Raman spectrum due to fluorescent emission from plasma proteins we are able to detect hemoglobin at concentrations from as low as 10-5 M and higher. The range of clinical relevance for hemolysis can be accurately resolved. The chemical analysis of liquid suspensions is of major interest to e.g. food industry, biotechnology and chemical industry. In this respect, it is desired to separate the suspending liquid from diluted particles, cells or beads [3]. A temporary particle or cell separation as proposed here is sufficient in order to analyze the suspending liquid in transit by micro-Raman spectroscopy. The microfluidic PDMS channels used in our whole blood experiments are 30-60μm wide and 40μm deep. In general, blood cells in flowing blood tend to migrate to the center of a microfluidic channel (Fahraeus–Lindqvist effect [4]), leaving a cell-free plasma region of 1-3μm at the channel walls. This cell-free plasma region can be locally expanded by the sudden enlargement of the microfluidic channel. In this way we create expanded semi-stagnant cell-free blood plasma regions of 5-20μm in width in close vicinity to whole blood flow. These regions are large enough to enable the application of localized confocal Raman spectroscopy exclusively in cell-free blood plasma. Hemolysis levels of whole bovine blood have been determined in this way. [1] M.D. Morris, D.J. Wallan, Anal. Chem., 51, 182–192 (1979) [2] T.C. Strekas and T.G. Spiro, Biochim. Biophys. Acta, 263, 830-833 (1972) [3] T. Kulrattanarak, R.G,M van der Sman, C.G. Schroen, R.M. Boom, Adv. Colloid Interface Sci, 142, 53-66 (2008) [4] R. Fahraeus, T. Lindqvist, The American Journal of Physiology, 96, 562–568 (1931)